ACS Publications. Most Trusted. Most Cited. Most Read
My Activity
Recently Viewed
You have not visited any articles yet, Please visit some articles to see contents here.
CONTENT TYPES

Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology

Cite this: J. Med. Chem. 2019, 62, 12, 5673–5724
Publication Date (Web):December 19, 2018
https://doi.org/10.1021/acs.jmedchem.8b01153
Copyright © 2018 American Chemical Society
Subscribed Access
Article Views
22592
Altmetric
-
Citations
LEARN ABOUT THESE METRICS
PDF (12 MB) OpenURL HONG KONG UNIV SCIENCE TECHLGY

Abstract

Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting noncatalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of noncatalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheads—beyond α,β-unsaturated amides—recently used in the design of targeted covalent ligands. Promising reactive groups that have not yet demonstrated their utility in TCI development are also highlighted. Special emphasis is placed on the discussion of reactivity and of case studies illustrating applications in medicinal chemistry and chemical biology.

1. Introduction

ARTICLE SECTIONS
Jump To

Covalent targeting has played a subordinate role in drug discovery for a long time. Although covalent modification of proteins(1,2) and nucleobases(3) is a key element in the regulation of biological systems, the systematic development of reactive drugs has been considered highly adventurous because of potential toxicity arising from promiscuous labeling, haptenization, and idiosyncratic drug reactions.(4,5) Nevertheless, over 40 covalent modifier drugs are currently approved by the FDA.(6) However, the mode of action of most of these compounds has been discovered serendipitously rather than resulting from rational design. Among the notable exceptions are inhibitors of amide-/ester-bond cleaving hydrolases, where reversible and irreversible covalent bond formation has long been used as a design strategy to address catalytic nucleophiles such as activated serine or cysteine residues.(7,8) During the past decade, covalent targeting has experienced a resurgence. So-called “targeted covalent inhibitors” (TCIs) addressing poorly conserved amino acids now provide the basis for a multitude of industrial drug discovery programs, especially in oncology.(5) According to the definition from a seminal review by Juswinder Singh and colleagues, a TCI is “an inhibitor bearing a bond-forming functional group of low reactivity that, following binding to the target protein, is positioned to react rapidly with a specific noncatalytic residue at the target site.”(5) Naturally, this concept can further be extended to other types of targeted covalent ligands beyond (enzyme) inhibitors. The renewed interest in covalent targeting is based on the perception that properly designed TCIs could offer multiple advantages, some of which would even result in a more favorable benefit–risk balance. Irreversible covalent inhibitors profit from nonequilibrium kinetics, potentially enabling full target occupancy, even if reversible binding affinity is moderate. In contrast to reversible binders, irreversibly bound compounds do not compete with natural ligands or substrates. Their durable target engagement can further decouple pharmacodynamics from pharmacokinetics provided that the protein of interest has a sufficiently slow rate of resynthesis.(6) The driving force and spatial requirements of covalent bond formation can be exploited to reduce the size of ligands without sacrificing potency and selectivity, thereby increasing “drug-likeness.” Considering that a substantial fraction of the drug candidates fail due to ADME issues,(9) the above features promise significant benefits. In cases where permanent protein modification is undesired, covalent-reversible chemistry can be used to harness potential advantages in terms of potency and selectivity while decreasing the risk of haptenization, inhibitor depletion by glutathione (GSH), and irreversible off-target modification.(10) The residence times of covalent-reversible inhibitors strongly depend on reversible interactions stabilizing the covalent complex. Thus, they can be tuned to fit the target’s turnover rate.(11) Ideally, the unmodified ligand would be released to re-engage with the target after protein degradation or after nonspecific covalent binding to off-targets incapable of stabilizing the covalent complex.
TCIs are typically generated by structure-based design from optimized reversible ligands, which are modified by attachment of an electrophilic covalent reactive group(12) (CRG; often termed “warhead”) to address a proximal amino acid, most frequently cysteine. Alternative strategies that have recently been employed to identify covalent binders include fragment-based(13−16) or tethering approaches(17) and DNA-encoded libraries featuring electrophilic ligands.(18,19) In TCI design, binding kinetics require special consideration. Because a detailed perspective on the implications of covalent binding kinetics has recently been provided,(20) only a brief overview shall be given at this place. TCI binding involves a two-step process in which an initial reversible binding event takes place, followed by the covalent bond-forming reaction. The first step is described by the binding constant KI, which accounts for the ligand concentration required to achieve a half-maximal rate of covalent modification. The second step is characterized by kinact, the maximal potential rate of covalent modification/inactivation. The overall efficiency of covalent target modification is best described as the second-order rate constant kinact/KI.(21) As a result, covalent inhibition is time-dependent and prolonged exposure leads to an increase in target occupancy. Consequently, KD, IC50, or EC50 values are not the appropriate measures for comparing covalent ligands because they vary over time and do not reflect the relative contribution of KI and kinact to the observed overall effect. Notably, time-dependent inhibition can also result from other factors like compound aggregation or instability in buffer and time-dependence cannot be considered as an ultimate proof of covalent binding despite the presence of a reactive group. On the other hand, very slow off-rate ligands may also be misinterpreted as covalent binders in washout and jump dilution experiments.(20) Beside the investigation of binding kinetics, cross-validation of the presumed covalent interaction, preferably by both mass spectrometry and X-ray crystallography, should thus be performed. Ideally, the latter data would be supported by studies with the mutated target protein devoid of the reactive amino acid and with congeneric ligands lacking the CRG. The two-step binding mechanism of TCIs differs from the one of reagents used for protein labeling or bioconjugation. The latter are used for chemo- but not site-selective covalent modification and preferably address the most reactive and accessible side chains. In this case, the reaction can be described by a one-step mechanism(20) without the requirement of specific reversible binding. Here, comparably reactive moieties relying solely on their intrinsic chemical reactivity toward the desired amino acids are used. Warheads to be utilized in TCIs require a more balanced reactivity profile. They should be reactive enough to form a covalent bond with the target residue in a proximity driven or templated manner, but only after the proper alignment of both reaction partners by the reversible binding event. Inherent reactivity should be reduced to a necessary minimum to prevent nonspecific off-target labeling or reaction with GSH. Because the reactivity of amino acid side chains varies as a function of their chemical environment, the reactivity of ideal CRGs should be readily adjustable to match the specific requirements of the target. Warheads intended for in vivo use, either in drugs or in chemical probes, should be chemically and (usually) metabolically stable and nontoxic. If chemical probes are intended for the investigation of specific targets or pathways in cells, some degree of chemical instability or toxicity might be tolerable. The latter class of ligands should be as selective as possible, which is not necessarily true for drugs. In contrast, reactive probes for activity-based protein profiling approaches (ABPP)(22) require a balanced amount of promiscuity to enable the profiling of a defined set of targets.
As a result of the time-dependent nature of covalent target engagement, even optimized compounds, which have demonstrated good selectivity in screening panels, can cause significant off-target labeling after extended exposure.(23,24) On the other hand, relatively small, reactive, and presumably unselective fragments can possess an unexpected degree of specificity when profiled against isolated proteins(15) or cellular proteomes.(13) The latter findings suggest that an increased reactivity might be exploited to achieve kinetic selectivity if the reactive moiety was completely neutralized by the target or metabolically deactivated after having engaged the target (vide infra). Certain highly reactive compounds such as α-cyanoacrylamide-derived Michael acceptors can further be useful for covalent-reversible targeting approaches.(25) Finally, the ideal balance of reversible binding affinity and specificity, inherent reactivity, and metabolic stability depends on the envisaged field of application and needs to be evaluated on a case by case basis.
There has been a very high interest in novel TCIs within the last years, the tremendous amount of published work having been compiled in a plethora of recent reviews.(5,6,26−39) Most of these reports focus primarily on noncatalytic cysteine residues addressed by α,β-unsaturated carbonyl compounds. Although cysteine-targeted Michael acceptors, most notably α,β-unsaturated amides, are clearly the dominant CRGs in the realm of current TCIs, they are not intended to be a key subject in this essay. An overview of α,β-unsaturated carbonyl compounds has already been provided in a recent Perspective article by Kay Brummond, Daniel Harki, and co-workers, extensively discussing the chemistry and reactivity of Michael acceptors as well as their application in drug discovery and beyond.(40) Instead, this Perspective focuses on less common warheads, recent innovations, and emerging concepts, highlighting case studies to illustrate their potential in medicinal chemistry and chemical biology. Reagents for protein modification, which have also been reviewed recently,(41−47) are not included except for certain functional groups where utility of the underlying reactivity in TCI design is anticipated. Likewise, warheads that have exclusively been used to target highly reactive active site nucleophiles, e.g., catalytic serine, threonine, or cysteine residues in proteolytic enzymes, are not a major focus of this Perspective and may be found elsewhere.(7,8,48) As an exception, few moieties engaging catalytic nucleophiles are discussed to highlight the underlying reactivity or design principles. It should further be pointed out that many warhead classes included in this article have previously found application in targeting the active sites of proteases and other hydrolase enzymes.
Despite the enormous amount of work that has been published on acrylamide-derived TCIs, systematic studies evaluating warheads beyond α,β-unsaturated amides are comparably sparse and scattered. Although we aimed to include all relevant and recent information, this work should rather be considered a (personally biased) perspective on interesting chemistry and current developments and not a comprehensive review of the field. This article covers literature published before July 2018, therefore interesting work which appeared during the revision of the manuscript (e.g., cysteine-targeted cyanamides as Janus kinase 3 inhibitors,(49) histidine-targeted linear alkyl bromides as 17β-hydroxysteroid dehydrogenase inhibitors,(50) and methionine-targeted epoxides as bromodomain inhibitors(51)), is not discussed. The article is organized by reactive amino acid side chains. Thus, warheads that have been used to target different residues may occur in several sections. (Pseudo)irreversible CRGs are discussed first, while covalent-reversible warheads may be found at the end of each section. A brief personal perspective on benefits and challenges of the respective CRG is given in each paragraph, and broader discussion may be found in the final Perspective section. Being a major subject of this article, inherent warhead reactivity and reaction rates are only compared directly if they were tested in the same assay. Otherwise, only a qualitative assessment is provided. Notably, there is still a heavy reliance on IC50/EC50 data in the field, although kinact/KI is the recommended measure for covalent binders. We have included kinetic data whenever it was available, but IC50 values are the basis for discussion if no such data has been reported.

2. Targeting the Cysteine Side Chain

ARTICLE SECTIONS
Jump To

Sulfur is the only third-row element encoded in proteinogenic amino acids and has a distinct role in biological systems. The thioether group in methionine and the neutral cysteine thiol group are only moderately nucleophilic. However, nucleophilicity is increased by several orders of magnitude for the thiolate form of the cysteine side chain, making it the strongest nucleophile among the 20 canonical amino acids.(52,53) Being highly polarizable, thiols and thiolates are soft bases according to Pearson’s hard and soft acids and bases (HSAB) concept.(54) The increased nucleophilicity and polarizability of thiolates compared to alkoxides is owed to the size and the high energy of the 3sp3 lone pairs. The thiol group can easily be deprotonated (pKa ≈ 8.6 for cysteine), and the pKa can shift several orders of magnitude within proteins.(55) While pKa values between 2.5 and 11.1 have been measured for cysteines’ thiols in proteins,(56) noncatalytic cysteine residues typically feature pKa values in the range between 7.4 and 9.1. Catalytic cysteines, in contrast, are pKa-perturbed to favor the highly nucleophilic thiolate anion.(55) Therefore, it is hardly surprising that cysteine has various roles in catalysis, e.g., in the active sites of cysteine proteases, ubiquitin ligases, or tyrosine phosphatases, but also in metal binding, structural stabilization (via the formation of disulfide bridges), and posttranslational/redox regulation.(57,58) Notwithstanding these key functions, cysteine is a relatively rare amino acid with a prevalence of only 1.9%.(59) Hence, noncatalytic cysteine residues played a key role in recent TCI design efforts. For example, all the six currently approved targeted covalent kinase inhibitors address an equivalently positioned, poorly conserved cysteine in the solvent-exposed front region of ErbB/HER family receptor tyrosine kinases or TEC family kinases like Bruton’s tyrosine kinase (BTK) via acrylic or butynoic amides.(35,60,61) An analogous cysteine has been used to generate highly selective Janus kinase (JAK) 3 inhibitors with a promising potential in the treatment of inflammatory disorders.(62) Besides TCIs, endogenous ligands such as cyclopentenone prostaglandins,(63) but also a multitude of other natural α,β-unsaturated carbonyl compounds,(40) covalently engage noncatalytic cysteines. As suggested by the differing pKa values, cysteine reactivity can vary over a wide range, and not all exposed cysteine residues may be readily amenable to covalent modification.(64) Targeting less reactive cysteine residues is not always straightforward, and success may crucially depend on precise positioning and sufficient reactivity of the CRG but also on assistance by the CRG or neighboring groups in deprotonating the thiol. Moreover, nonessential cysteines are prone to mutation (see for example the EGFR C797S resistance mutant)(65) which can be considered an Achilles’ heel of cysteine-targeted covalent drugs, especially in oncology. On the other hand, acquired cysteine residues (e.g., in the oncogenic K-Ras G12C or the p53 Y220C mutants) may open up new avenues for addressing challenging targets.

2.1. Cysteine Addition to Metabolically Labile Monomethyl Fumarates

Although targeted covalent inhibitors derived from α,β-unsaturated amides are not a major topic here, some recent “noncanonical” approaches merit further discussion. As mentioned, TCIs often feature excellent selectivity for their targets upon short-term treatment but extended exposure has been shown to erode selectivity.(23,66) Slower covalent modification of off-targets can be missed by classical screening techniques typically investigating short-term effects (minutes to few hours). A possible solution to this issue is provided by covalent-reversible binders, such as the above-mentioned α-cyanoacrylamides. Another interesting approach to minimize time-dependent off-target modification has recently been developed in Benjamin Cravatt’s laboratories. While investigating the cysteine labeling profile of the immunomodulatory drug dimethyl fumarate (DMF) in T cell proteomes, they found the active metabolite monomethyl fumarate (MMF) to be devoid of substantial cysteine reactivity,(67) a finding that can be attributed to the decreased intrinsic reactivity of monomethyl fumarate but that might also reflect the presumably lower cell permeability of MMF. On the basis of this observation, the hypothesis arose that replacement of a conventional α,β-unsaturated amide in a cysteine-targeted inhibitor by a methyl fumarate residue could furnish compounds that rapidly react with their targets while the slower inactivation by esterases prevents off-target modification (Figure 1).

Figure 1

Figure 1. Kinetic selectivity of fumaric acid esters. Selectivity is povided by rapid bond formation with the target. Slightly slower ester cleavage deactivates the warhead, preventing even slower labeling of undesired proteins.

To test this hypothesis, the approved covalent Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib(68) (1, Figure 2), which is known to react with off-targets in a time-dependent manner,(23) was chosen for a first proof of concept study. Ibrutinib features a piperidin-3-yl-linked acrylamide warhead that labels BTK at Cys481.(69,70) The latter was replaced by several amide-linked fumarates (37). Some of the studied compounds (2 and 57) were further equipped with an alkyne to provide a chemical handle enabling the attachment of tags for enrichment and target identification. Both ibrutinib-derived probe 2 and its methyl fumarate analogue 5 labeled BTK in Ramos cell lysates. As expected, fumarate 5 reacted faster with cysteine and possessed the higher reactivity toward the cellular proteome. When incubated with HEK293T cells stably expressing human carboxylesterase (hCES) 1, ibrutinib remained unaffected while the methylfumarate-derived inhibitor 3 was rapidly converted to the unreactive acid 4. This effect was not observed when using HEK293T cells stably expressing methionine aminopeptidase (MetAP) 2 as a control. A significant reduction in time-dependent proteome labeling accompanied by a modest decrease in BTK activity (ca. 10-fold) was observed for probe 5 when incubated over 24 h with a 6:1 coculture of Ramos and HEK293T cell lines, which was supposed to mimic the hCES1 activity in tumor xenografts. In contrast, the labeling profile of the ibrutinib-derived probe 2 remained unaffected under these conditions. Competition experiments employing gel-based or MS proteomics(71) confirmed that BTK engagement by inhibitor 3 was only marginally affected by hCES1, supporting the notion that kinase modification occurs at a higher rate than enzymatic ester hydrolysis. Only BTK and the kinase TEC, sharing an equivalently positioned cysteine,(62) reacted with 3 in a hCES1-insensitive manner while 1 retained its proteomic profile in the presence of the esterase.

Figure 2

Figure 2. Ibrutinib-derived fumarate esters and analogous probes equipped with a click handle.

Subsequent profiling of probe 5 and its isopropyl ester analogue 6 in rodents (20 mg/kg ip) demonstrated BTK engagement in vivo, although both compounds were poorly stable in mouse plasma (t1/2 = < 2 min). Pretreatment with the covalent CES inhibitor JZL184,(72) however, increased the plasma stability to 25.5 and 352 min for 5 and 6. In contrast, probe 2 and the free acid 7 did not require CES blockage to obtain reasonable plasma stability (t1/2 = 168 and 129 min). Both 5 and 6 demonstrated significantly reduced off-target labeling in different tissues while substantial reactivity toward BTK was maintained. Although these results are qualitative in nature, they underline the increased BTK selectivity of the fumaric acid esters in vivo.
Although the metabolic inactivation of the reactive species to promote kinetic selectivity is a promising concept, the generalizability of this approach remains to be shown. Alternative esters might be required to fine-tune stability against esterases, and those will need to fit the respective binding pockets. As suggested by the authors of the above study, alternative warheads with limited metabolic stability such as acrylates or thioacrylates may have the potential to address these issues.(24) However, optimization of such compounds for clinical use, especially when considering oral administration will further complicate the situation due to the prolonged and more complex kinetics of uptake and distribution as well as first-pass metabolism. Moreover, proteins with a high turnover constitute a challenge to this conceptual framework as they require sustained exposure for pharmacological efficacy. Nevertheless, this study demonstrates that kinetic selectivity is achievable with metabolically labile warheads and it will be interesting to see whether related concepts will expand our current toolbox for TCI design.

2.2. Cysteine Addition to Allenamides

Recently, allenamides have been suggested as bioisosteres of α,β-unsaturated amides for protein labeling purposes and inhibitor design. Although the reactivity of allenamides (general structure 8, Figure 3) toward thiols has been known for a long time,(73,74) Teck-Peng Loh and colleagues were the first to investigate these reagents for cysteine labeling applications.(75) They found a high intrinsic reactivity toward the thiol(ate) of isolated cysteine as well as for terminal and internal cysteine residues in peptides and proteins at pH 8. However, neither were reaction rates determined nor was a qualitative comparison of reactivity between aniline-derived allenamides and alkylamine-derived analogues provided. No labeling of other nucleophilic amino acids was observed. Upon reaction with cysteine at the sp-hybridized carbon center, only the thermodynamically disfavored nonconjugated product 10a was formed, which is probably due to prevalence of the mesomeric structure 9a favoring protonation in the α-position. Notably, a similar regioselectivity was recently observed for selenocysteine conjugation.(76) Compared to their keto and ester analogues, terminal allenamides have a much lower tendency to undergo [3 + 2] cycloaddition reactions and cysteine labeling was shown to be irreversible even in the presence of a 100-fold excess of glutathione or dithiothreitol (DTT).

Figure 3

Figure 3. Mechanism of cysteine addition to allenamides. The prevalence of the mesomeric structure 9a rationalizes the formation of the nonconjugated product. An alternative mechanism involving attack of the neutral thiol to form a zwitterionic species followed by proton transfer was proposed by Loh and co-workers.

While the Loh group only tested this CRG for protein labeling, a subsequent study by Yujun Zhao and colleagues exploited the allenamide moiety as a bioisosteric replacement of the acrylamide functionality in the approved EGFR inhibitor osimertinib (11, Figure 4).(77) The allenamide warhead was well tolerated in terms of potency, and analogue 12 was slightly more active in blocking the EGFR T790M/L858R mutant compared to the parent compound (IC50 = 1.4 nM vs 3.9 nM for EGFRT790M/L858R). However, the compound partially eroded osimertinib’s selectivity over the wild-type enzyme (IC50 = 28 nM vs 142 nM for EGFRwt). Further optimization furnished low nanomolar inhibitors with increased (up to 43-fold for compound 13) or decreased (only 6-fold for compound 14) selectivity over the wild-type enzyme. Selectivity in the enzyme assay, however, did not translate into cellular selectivity for NCI-H1975 (EGFRL858R/T790M) versus A549 (EGFRwt) cells. Target engagement in NCI-H1975 cells was suggested by Western blot, but neither was covalent binding unambiguously demonstrated nor were off-targets assessed. Allenamide 12 showed poor oral exposure in mice, and both 12 and 14 possessed an over 10-fold lower stability in fetal bovine serum compared to osimertinib. The decreased stability is likely to result from the higher intrinsic reactivity of the allenamide warhead compared to osimertinib’s acrylamide. Pseudo-first-order rates for the reaction of compound 14 with glutathione (k′ = 0.303 min–1) were shown to be 7–28-fold higher than for the approved acrylamide-derived kinase inhibitors afatinib (k′ = 0.044 min–1), osimertinib (k′ = 0.011 min–1), and ibrutinib (k′ = 0.011 min–1). Although terminal allenamides offer certain advantages such as good accessibility, irreversibility, compact size, and a well-defined geometry, their utility in medicinal chemistry remains thus limited by the high intrinsic reactivity. In analogy to α,β-unsaturated amides,(12) a decrease in reactivity might be achieved by employing alkylamine-derived amides. In certain cases, for example when poorly nucleophilic cysteines(78) need to be addressed, the higher reactivity may provide a benefit, assuming that the reversibly binding part of the inhibitor confers sufficient kinetic selectivity to minimize off-target labeling.

Figure 4

Figure 4. Osimertinib-derived allenamides as EGFR inhibitors.

2.3. Cysteine Addition to Linear 3-Aryl and Alkyl Propiolonitriles

Alain Wagner and co-workers have recently established 3-aryl propiolonitriles (general structure 15, Figure 5A) as reagents for selective cysteine modification in peptide mixtures.(79) The obtained vinyl thioether adducts were stable against thiols over a wide pH range (from 0–14), reducing agents (tris(2-carboxyethyl)phosphine (TCEP) and DTT), and a reasonable stability was also demonstrated in plasma and living cells. In contrast, structurally related aryl alkynones undergo thiol exchange via addition of a second thiol and subsequent elimination of one thiolate (Figure 5B).(80) Reactivity of unsubstituted 15 is presumably too high for medicinal chemistry applications. However, reactivity could be decreased by substituents with a +M effect in the para-position (and vice versa, with substituents featuring a −M effect) and dropped substantially when ortho-substituents were introduced. Moreover, the analogous 3-cyclohexylpropiolonitrile (16) reacted about 10 times slower compared to unsubstituted 15, however, the obtained reaction product also proved to be slightly less stable. Although no applications in a medicinal chemistry or chemical biology context have been discussed so far, this linear electrophile could become very useful when precise and rigid spatial placement of the warhead is required (provided that the binding cavity can host this elongate functionality). Still, it remains to be seen if this CRG can be attenuated to an appropriate intrinsic reactivity to become useful for chemical probe development.

Figure 5

Figure 5. Propiolonitriles as potential TCI warheads: (A) 3-Aryl and 3-alkyl propiolonitriles. (B) Mechanism of cysteine addition and thiol exchange.

2.4. Cysteine Addition to Alkenyl or Alkynyl-Substituted Heteroarenes

Electron-deficient heteroaryl groups with (substituted) vinyl or ethynyl residues constitute a structural motif frequently found in patent applications claiming covalent inhibitors. These moieties are supposed to react with thiols via a conjugate addition, in which the intermediate negative charge is stabilized by the heteroarene. This reaction type has been known for a long time. For example, 4-vinylpyridins have been used to modify cysteines since the 1960s(81,82) and chemical reactions exploiting alkenylated or alkynylated aromatic heterocycles for nucleophilic additions are not uncommon (e.g., refs (83−88)). Vinylpyridines have also been applied in cysteine-specific protein labeling.(89,90) and their reactivity can be readily modulated.(91) In this light, it is surprising that only a few and relatively limited systematic studies evaluating this chemistry for cysteine-targeted inhibitors have been published so far.
An early isolated series of publications on the application of such CRGs in medicinal chemistry was reported by David Uehling and co-workers from GSK in 2008/2009.(92) They serendipitously found a noncatalytic cysteine of ErbB kinases to react with ethynylthienopyrimidine-based inhibitors. Although the ethynyl moieties were attached to the thiophene ring, which is usually electron-rich, covalent bond formation with Cys803 in ErbB4 and Cys797 in EGFR was observed. In a subsequent SAR study, the influence of a terminal substituent at the ethynyl moiety was evaluated for both a 6-ethynylthieno[3,2-d]pyrimidine (1720, Figure 6A) and a 6-ethynylthieno[2,3-d]pyrimidine series (exemplified by compound 21). Consistent with a mechanism in which the thiol(ate) attacks the triple bond generating a transient negative charge, the 6-ethynylthieno[3,2-d]pyrimidine-derived compounds were more reactive due to better resonance stabilization of the latter. However, labeling was generally slow. While the acrylamide-derived control compound canertinib (CI-1033)(93) completely labeled EGFR within less than 3 h, only 67% of covalent modification was observed for the terminal alkyne 17 after 20 h as determined by MS. Interestingly, the attachment of a 2-(R)-pyrrolidine residue (18) significantly accelerated covalent inactivation, leading to 83% labeling after 3 h and full labeling after 20 h, respectively. This acceleration was neither observed with an analogous compound featuring a (R)-2-aminoethyl substituent (19) nor with a weakly basic 2-pyrazine derivative (20). The rate-enhancing effect of methylene-linked amino groups has been described previously for propiolamide- and acrylamide-derived EGFR inhibitors.(94,95) In the suggested mechanism, the basic amine increases the nucleophilicity of the thiol group by hydrogen bonding/deprotonation (Figure 6B). The distinct effect of the (R)-pyrrolidine residue compared to the primary propargylic amine in 19 might be rationalized by the spatial requirements for proton abstraction. An X-ray crystal structure confirmed covalent binding of compound 17 to Cys803 in ErbB4 (Figure 7) while EGFR labeling at Cys797 was proven by MS. Key compound 18, a potent EGFR, ErbB2, and ErbB4 inhibitor (IC50 = 7, 13, and 66 nM, respectively) with cellular IC50 values in the midnanomolar range featured >100-fold selectivity against 30 from 31 kinases in a small panel. Remarkably, it did not inhibit interleukin-2-inducible T-cell kinase (ITK) possessing an equivalently positioned cysteine. Dosed orally at 30 and 100 mg/kg b.i.d., compound 18 was an effective inhibitor of tumor growth in a murine BT474 cell xenograft model while compound 19 showed slightly lower activity in the same model system.

Figure 6

Figure 6. 6-Ethynylthienopyrimidine as covalent ErbB kinase inhibitors. (A) 6-Ethynylthieno[3,2-d]pyrimidine and 6-ethynylthieno[2,3-d]pyrimidine-derived inhibitors. (B) Suggested mechanism of cysteine addition.

Figure 7

Figure 7. X-ray crystal structure of compound 17 in complex with the ErbB4 kinase domain (PDB 2R4B). The terminal alkyne moiety has reacted with Cys803 to form a vinyl thioether adduct. The N1-atom of the pyrimidine ring is further anchored to the backbone NH of Met799 in the hinge region via a hydrogen bond. The pyrimidine N3-atom is engaged in a water-mediated hydrogen bond to the side chain of Thr860 preceding the conserved DFG motif.

In several follow-up studies, the same team investigated the effects of substituents at the 4-position of the pyrrolidine ring,(96,97) the stereochemistry(97) at the pyrrolidine substituent, and modifications at the aniline headgroup.(98) Although some improvements of ADMET properties were achieved, no major advances in terms of biochemical and cellular potency and covalent labeling efficiency could be made. Moreover, it should be pointed out that neither biochemical nor cellular inhibitory potencies showed a clear correlation with the propensity to covalently modify the target in vitro, accentuating that the comparatively slow cysteine addition was not the key driver of biological activity. Since no further studies with this inhibitor class have been reported since 2009, it can be assumed that their development was discontinued. Nevertheless, the provided data shows that heteroarene-derived Michael-type acceptors are compatible with a physiological environment and can in principle be used as CRGs in drug discovery. Systematic studies, however, will be required to explore the tunability and versatility of this type of warhead. Moreover, when alkynylated heteroaryl groups are employed as acceptors, the stability of the adducts toward a second thioether addition and accompanying thiol exchange reactions (see section 2.3) will necessitate further investigation.
More recently, the group of Rob Leurs modified reversible 2-aminopyrimidine-based antagonists of the human histamine H4 receptor with a 2-vinyl substituent to covalently address Cys98 located in the transmembrane domain (TM) 3.(99) Both VUF14480 (22, Figure 8), the key compound of this study, and its nonelectrophilic analogue VUF14481 (23), bound the hH4 receptor with similar (apparent) affinities in the upper nanomolar range. Both compounds acted as partial agonists and potencies in a GTPγS-binding assay were comparable to the respective affinities but time-dependence was not determined in either assay system. As expected, 22 reacted with GSH and cysteine ethyl ester to form covalent adducts, while 23 was unreactive. Unfortunately, only indirect evidence for the covalent engagement of Cys98 was provided and no validation by MS or X-ray crystallography performed. To this end, the authors showed that only 22, but not 23, decreased [H3]-histamine binding after washout, an effect that was not observed when performing the same experiment with the hH4 C98S mutant. However, even at the highest concentration of 10 μM (incubation for 1 h), 22 only partially blocked [H3]-histamine binding after washout, indicating a slow reaction and incomplete covalent labeling. Interestingly, the affinities of both compounds did not significantly differ between the wild-type receptor and the C98S mutant, suggesting reversible binding to be the main contributor to the latter.

Figure 8

Figure 8. 2-Vinylpyrimidine-derived H4 receptor ligand VUF14480 and the unreactive analogue VUF14481.

2.5. Cysteine Addition to Nonactivated Terminal Alkynes

Propargylamines such as selegiline and rasagiline are well-known as mechanism-based covalent monoamine oxidase (MAO)-B inhibitors in the treatment of Parkinson’s disease,(100,101) and terminal alkynes have further found application as activity-based probes for CYP enzymes.(102,103) In these compounds, however, the respective alkynes undergo oxidative activation to generate electrophiles, which can covalently engage their protein targets. While strain activated alkynes are known to react with cysteine residues,(104,105) the nonactivated terminal alkynes employed in CuAAC click reactions(106) have been assumed to be inert in biological samples. In this light, recent reports on protein-templated cysteine addition to nonactivated, and thus poorly electrophilic, terminal alkynes are quite remarkable (exemplified by general structure 24, Figure 9A). In 2013, the groups of Huib Ovaa(107) and Henning Mootz(108) independently discovered this reactivity while employing propargylamide-labeled ubiquitin (Ub), SUMO, or peptides derived thereof, intended for click-chemistry-based labeling. The active site cysteine of deubiquitinating isopeptidases (DUBs) and SUMO proteases selectively attacked the triple bond at the 2-position forming the Markovnikov vinyl thioether adduct. Interestingly, N-but-3-ynyl amide (24a) and even N-hex-5-ynyl amide homologues (24b) were also reactive while the allylic amide 24c showed poor reactivity (Figure 9B). Introduction of a terminal methyl group (24d) or geminal 2,2-dimethyl substitution (24e) at the propargyl residue prevented cysteine addition.(107) The approach could also be applied to other cysteine proteases such as caspase-1(107) or more recently the viral leader protease Lbpro.(109) Although these results partially question the biological inertness of terminal alkynes used in click-chemistry labeling applications, the underlying reaction seems to be quite specific and, so far, limited to certain cysteine proteases. It has been proposed that this reaction requires the stabilization of the intermediary carbanion by the hydrogen bond donors in the oxyanion hole.(110) In the SUMO-protease Senp1, however, mutation of key residues in the catalytic triad (i.e., H533A and D550A) and the oxyanion hole (Q597A) did not prevent the formation of the covalent adduct.(108) Further mechanistic investigations will be required to elucidate the structural requirements promoting these reactions and it remains to be seen, if noncatalytic cysteines could covalently trap propargylamides and similar terminal alkynes in certain settings.

Figure 9

Figure 9. Nonactivated terminal alkynes as cysteine traps. (A) Reaction of C-terminally propargylated ubiquitin 24 with the active site cysteine in DUBs. (B) Reactive (top) and nonreactive (bottom) analogues.

2.6. Cysteine Targeting by Nucleophilic Aromatic Substitution (SNAr) Reactions

Although nucleophilic aromatic substitution (SNAr) reactions have a long history in covalent protein targeting,(111) they are still largely underdeveloped in the field of TCIs. In this type of reaction, covalent labeling is effected by a nucleophile, e.g., a cysteine thiol(ate), displacing a leaving group from an electron-deficient aryl ring. Classically, SNAr reactions have been considered to proceed via a stepwise addition–elimination mechanism (aryne, radical and SN1-type mechanisms are not considered here) involving negatively charged intermediates termed Meisenheimer- or σ-complexes, which are stabilized by the electron-withdrawing group(s) (Figure 10A).(112,113) More recent studies suggest that many SNAr-reactions rather proceed in a concerted manner via Meisenheimer-like transition states.(114) The reactivity of (hetero)arenes in SNAr reactions increases with the electron deficiency of the aromatic system. Reactivity can be readily increased by adding electron-withdrawing (typically −M) substituents or heteroatoms, which are ideally positioned to the ortho- or para-position of the leaving group. In most cases, the rate of SNAr-reactions is determined by the attack of the nucleophile while elimination of the leaving group is faster.(113) Therefore, the SNAr reactivity of aryl halides typically ranks F ≫ Cl ≈ Br > I, roughly correlating with the increasing polarization of the Cδ+–Xδ− bond. However, different rankings might be observed because the loss of the nucleofuge can also become rate limiting under certain conditions. A detailed discussion of all factors influencing SNAr reactions is beyond the scope of this article and can be found elsewhere.(113) Yet, it is important to note that the reactivity of SNAr electrophiles can be readily adjusted by varying the electronic nature of substituents, and the leaving group.

Figure 10

Figure 10. SNAr-based covalent ligands. (A) Classical mechanism of the SNAr reaction with cystein. (B) Selected examples of early SNAr-based ligands and reagents. Leaving groups are highlighted in red.

In early applications, SNAr warheads have been exploited in inhibitors like the 2-chloroquinoxaline L-764406 (25, Figure 10B),(115) the 2-chlorobenzamide GW9662 (26)(116) or the 2-sulfonylpyridine GSK3787 (27),(117) which target peroxisome proliferator-activated receptors (PPARs) by labeling different noncatalytic cysteines. 2-Sulfonyloxadiazoles, as exemplified by 28, have been used to covalently block the catalytic serine of AmpC β-lactamases(118) or as maleimide replacements for the generation of serum-stable antibody conjugates.(119) Other heteroarylsulfone-derived SNAr reagents such as MSBT (2-(methanesulfonyl)benzothiazole, 29)(120) have been employed as cysteine capping reagents and for protein conjugation.(121) The SNAr reactivities of perfluoroarenes(122,123) and dichloro-s-tetrazine(124) have been further exploited for peptide stapling. Notably, the cysteine reactivity of highly activated SNAr electrophiles has been correlated with their skin sensitization potential.(125)
In 2011, a team of researchers around Kiplin Guy identified methylsulfonyl nitrobenzoates exemplified by MLS000389544 (30) as low micromolar irreversible inhibitors of the thyroid hormone receptor (TR) β/steroid coactivator (SRC) 2 interaction by high throughput screening (HTS) of a library of 500000 compounds.(126) These compounds selectively labeled Cys298 as determined by MS, the same cysteine that had previously been shown to react with Michael acceptors formed in situ from β-aminoketones.(127) It is noteworthy that nucleophilic displacement of the methylsulfonyl residue was selective in the proximity of an activated ester moiety as an alternative reaction site. As one would expect, removal of the nitro group resulting in a less electrophilic analogue was detrimental to activity. However, the replacement of the methylsulfonyl group by chloride or fluoride also furnished poorly active compounds, which is more surprising because sulfones are typically less reactive than fluorides in SNAr reactions.(113) This finding might reflect the involvement of the methylsulfonyl residue in reversible binding or point to the complex interplay of different factors on SNAr reactivity.
In 2015, John Kuriyan and co-workers reported the sulfonyltetrazole 31 (Figure 11), the NBD-dye 32, and the sulfonylpyrimidine carboxamide 33 as low molecular weight inhibitors antagonizing the interaction between the tandem Src homology (SH) 2 domains of the nonreceptor tyrosine kinases ZAP-70 and Syk and doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs).(128,129) These compounds, which were identified by HTS, inhibited the SH2–ITAM interaction at low micromolar concentrations in a time-dependent manner. X-ray and MS experiments confirmed the specific binding to single cysteines when no excess of the reagents was used. The X-ray structures of compounds 31 (PDB 4XZ0) and 32 (PDB 4XZ1) in complex with the ZAP-tSH2 domain did not show any specific interactions with the protein and switching from TCEP- to DTT-containing buffer completely abolished inhibition by all three compounds. Therefore, it can be assumed that the high intrinsic reactivity of these electrophiles precludes application in cells or in vivo.

Figure 11

Figure 11. Covalent inhibitors antagonizing the interaction of ZAP-70 and Syk with ITAMs. Leaving groups are highlighted in red.

A reactivity analysis of electron-deficient halogenated (hetero)aryl fragments in cellular proteomes was reported in 2014 by Eranthie Weerapana and co-workers.(130) By using an alkyne-tagged series of chloronitrobenzoic amides (general structure 34, Figure 12A), they found that selectivity critically depends on the positioning of the nitro group. As expected, a meta relationship between the nitro moiety and chloride leaving group gave an unreactive compound, whereas compound 34a (Figure 12B) with the nitro group in the para-position was the most reactive. Dichloropyridines and -pyrimidines (general structure 35) also showed limited reactivity, while an analogous triazine (35a) caused more promiscuous labeling reflecting the highly electron-deficient nature of this heteroarene. Intriguingly, however, 34a and 35a affected distinct sets of proteins, and peptide mapping showed that 34a preferentially labeled cysteine thiols while 35a had an unexpected preference for the lysine side chain. Although this behavior could not be fully rationalized, complementary experiments showed that 35a preferentially reacts with cysteine in solution, emphasizing that solution reactivity is not necessarily predictive of reactivity within protein binding sites.

Figure 12

Figure 12. Electron-deficient (hetero)aryl probes used for evaluation of SNAr-based labeling in proteomes. (A) General structures. (B) Selected compounds preferably labeling cysteine or lysine.

A recent study from Walter Fast and co-workers investigated 4-halopyridines as quiescent SNAr electrophiles.(131) They found protonation of the pyridine nitrogen atom to be a critical factor for the reactivity of this compound class. To this end, the reaction with GSH at physiological pH was compared between 4-chloropyridine (36, Figure 13A) and its charged N-methylated analogue (37, Figure 13B). Earlier data from Flanagan et al. had already demonstrated that 2-chloropyridine reacts only slowly with GSH at pH 7.4, with a rate constant in the same range as observed for N-methylacrylamide.(12) Similarly, 36 showed very low reactivity comparable to that of ampicillin, styrene oxide, and acrylamide. In contrast, the N-methylated analogue’s reactivity increased by more than 3 orders of magnitude, thus being in the same range as the one of iodoacetamide. Arguing with the classical SNAr mechanism, the accelerating effect would be attributed to better stabilization of the intermediate adduct being a neutral dihydropyridine instead of a negatively charged σ-complex in this case (Figure 13B). Against the initial expectation that 4-chloropyridine would be more reactive than the respective bromo and iodo-derivative, reaction rates with thiophenol increased in the order Br > I > Cl (4-fluoropyridines were not included in this study). The same ranking was obtained when the 4-halopyridines were assayed for their capability of inactivating dimethylarginine dimethylaminohydrolase (DDAH) 1 by reacting with the catalytic cysteine residue. For further investigations, a series of disubstituted 4-halopyridines with various substituents at ortho- or meta-position was prepared (general structure 38, Figure 13C). All of these compounds showed no or little reactivity in the GSH assay, even if an electron-withdrawing substituent was present in the C3-position. Profiling this structurally heterogeneous series against DDAH revealed no clear correlation between kinact/KI and the substitution pattern. However, in a set of 2-methylpyridines with an increasing number of fluorine substituents at the methyl group (pKa values ranging from 4.7 to 0.4), a correlation between the pKa of the protonated pyridine and reactivity was observed. Counterintuitively, more electron-deficient compounds reacted more slowly. This suggests that the DDAH enzyme, which possesses a proximal aspartate (Asp66), would stabilize the protonated pyridine, thereby enhancing its reactivity. However, no X-ray crystal structures or mutation studies were provided to fortify this hypothesis. Further experiments were performed with alkyne-tagged probes 39 and 40 (Figure 13D) in soluble Escherichia coli proteomes. Gel-based analysis showed that 39 only modifies a few proteins and the number even decreases when cell lysates are denatured prior to exposure. In contrast, N-methylated analogue 40 labeled much more proteins and the number increased substantially in denatured lysates. These observations suggest that the neutral 4-halopyridines require activation by the protein environment for becoming reactive, while the charged N-methyl-4-halopyridines can promiscuously label surface-exposed nucleophiles, which are more abundant in the denatured samples.

Figure 13

Figure 13. 4-Halopyridines as quiescent SNAr electrophiles. (A) SNAr-reaction with 4-chloropyridine according to the classical mechanism. An anionic Meisenheimer intermediate is formed. (B) Analogous mechanism of the reaction with N-methyl-4-chloropyridine. A neutral dihydropyridine species is formed as the intermediate. (C) General structure of the investigated compounds. (D) Alkyne-tagged probes used for proteomic analysis.

In a recently published drug discovery campaign, Robin Fairhurst and co-workers from Novartis aimed to identify covalent inhibitors targeting a rare cysteine (Cys552)(35) located in the hinge region of the fibroblast growth factor receptor (FGFR) 4 tyrosine kinase.(132) A high throughput screen revealed compound 41 (Figure 14A) as a nanomolar inhibitor (IC50 = 32 nM) of wild-type FGFR4 sparing the FGFR4 C552A mutant. The compound did not hit the other FGFR family members (FGFR1–3), which are devoid of this cysteine. The high activity of this structurally simple molecule can be attributed to displacement of the 6-pyridyl chlorine atom by the cysteine’s thiol. Covalent modification at Cys552 was confirmed by MS and X-ray crystallography (Figure 14B). The compound possessed a 170-fold lower rate constant (kinact/KI = 3.0 × 104 M–1s–1) than an acrylamide-based inhibitor from the same study targeting Cys477 common to all FGFR family members. Although 41 selectively labeled Cys552 under the conditions used in MS and X-ray experiments, a 10-fold excess also modified Cys477 indicating at least some promiscuity. Interestingly, the latter modification seems to leave kinase activity unaffected, highlighting the fact that enzymatic or competition-based kinase assays are typically not suited for the identification of nonactive site binders. Although the authors recognized the potential of this compound for optimization in terms of reactivity, potency, and selectivity, the inhibitor was dropped in favor of a covalent-reversibly binding aldehyde (vide infra, section 2.12) owing to the short FGFR4 resynthesis half-life (<2 h).

Figure 14

Figure 14. (A) Structure of the covalent FGFR4 inhibitor 41. (B) X-ray cystal structure of 41 in complex with the FGFR4 kinase domain (PDB 5NUD). Both pyridine rings form a hydrogen bond with the backbone NH group of Ala553, while the nitro group stabilizes the active conformation via an intramolecular hydrogen bond with the diarylamino NH. The covalent bond with Cys552 is formed by SNAr displacement of the 6-chloro group from the 2-amino-3-nitropyridine moiety. A weak water mediated H-bond between the nitro substituent and the Arg483 guanidinium group was omitted for clarity.

Weijie Hou and co-workers reported in early 2018 on putatively covalent EGFR inhibitors featuring SNAr warheads to address Cys797.(133) Their rationale was the replacement of the α,β-unsaturated amide found in afatinib and analogous compounds by an electron-deficient aryl ring equipped with a halide leaving group (general structure 42, Figure 15). Several potent inhibitors with low nanomolar affinities were identified, but activity differences between electrophilic and nonelectrophilic compounds were only moderate. Covalent modification was not directly assessed, but washout experiments with key compound 42a indicated only reversible binding although the inhibitor reacted with GSH confirming its inherent reactivity. As the EGFR is known to react with various acrylamide-derived inhibitors,(134) it can be assumed that inappropriate positioning of the electrophile prevents covalent trapping in this case.

Figure 15

Figure 15. EGFR inhibitors with SNAr warheads which do not form the predicted covalent bond with Cys797.

Although the above studies show that targeted covalent inhibition with SNAr warheads is feasible, no substantial efforts on the (successful) compound optimization were reported nor were the compounds profiled in a broader array of assays. A more comprehensive medicinal chemistry study on verifiable SNAr-based irreversible inhibitors was published by Kevin Chen and colleagues at Merck Sharp & Dohme.(135) The researchers optimized a screening hit based on an indole core (43, Figure 16) to inhibit the hepatitis C virus (HCV) NS5B polymerase. While optimizing the indole N1-substituent, the introduction of a nitrile group ortho to the fluoride at a 2-fluoro-5-methylsulfonyl benzyl residue (45) was serendipitously found to promote a 100-fold improvement in activity in a cellular replicon assay. As the parent compound 44 was known to bind to the “palm” site of the NS5B protein, covalent bond formation with the noncatalytic Cys366, a residue that had previously been targeted with benzylidene rhodanine-derived covalent-reversible inhibitors,(136) was assumed to be responsible for this unexpected leap in activity. A variety of analogues with different electron-withdrawing substituents and leaving groups at the phenyl ring was prepared, but no electron-deficient heteroarenes had been explored at that stage. While most modifications gave only weak to moderately active compounds, a strongly electron-withdrawing nitro group in the para-position of the halide leaving group (46 and 47) was able to promote excellent biochemical and cellular activity. Notably, binding kinetics were not investigated. Further optimization yielded key compound 48, which exhibited a good PK profile in monkeys and dogs. The inhibitor featured an oral bioavailability of 30% and 95%, an AUC (po, 3 mg/kg) of 3.4 and 11 μM·h, and a half-life of 1.3 and 1.2 h, respectively, in the aforementioned species. The X-ray crystal structure of 48 in complex with the HCV NS5B polymerase (PDB 3TYQ) as well as MS experiments unambiguously confirmed the expected covalent binding mode. An NMR-based method (ALARM-NMR)(137) exploiting the high nucleophilicity of cysteines in human La antigen showed no promiscuous thiol reactivity. Moreover, the compound was found to be clean in kinase and protease panels. Although the nitro group was regarded with skepticism due to known potential safety issues caused by partial reduction products,(138) no toxicity could be observed in a Salmonella/mammalian microsome mini-AMES assay, an in vitro micronucleus assay and in single-dose animal PK studies.

Figure 16

Figure 16. Development of covalent HCV NS5B polymerase inhibitors with SNAr warheads.

In a follow-up study, the same team evaluated heterocyclic substitutes for the 2-fluoro-5-nitrobenzyl residue.(139) Although the nitro group did not promote any obvious toxicity in the above study, it was considered to be safer to avoid this functionality. An initial meta-linked 2-chloropyridine series exemplified by 49 (Figure 17A) gave acceptable potencies in the biochemical assay, however, cellular EC50 values increased to the low micromolar range. As it was shown later, these moieties were not capable of covalent target engagement. Replacement of the pyridine ring by a 2-chloroquinoline moiety rescued cellular activity. Although small substituents in the C6- and C7-position of the quinoline ring were tolerated, the best results were obtained with an undecorated 2-chloroquinoline moiety. Key compound 50 featured low nanomolar activities in both assays. Covalent binding to Cys366 was proven by MS and X-ray crystallography (Figure 17B), and no labeling of any of the enzyme’s other 21 cysteines was detected. Intriguingly, the compound was stable toward harsh chemical conditions such as refluxing in aqueous lithium hydroxide or 0.5 M ammonia/dioxane pointing out the low intrinsic reactivity of the warhead. The compound possessed a similar clean profile as 48, showed no significant CYP or hERG interactions, only limited toxicity up to 2000 mg/kg in rats, and favorable in vivo drug metabolism and pharmacokinetic (DMPK) properties in rat, dog, and monkey. Compound 50 was thus suggested as a candidate for further development.

Figure 17

Figure 17. Second generation NS5B polymerase inhibitors with SNAr warheads. (A) Reversibly binding 2-chloropyridine 49 and the irreversibly binding quinoline analogue 50. (B) X-ray crystal structure of key compound 50 covalently bound to Cys366 of HCV NS5B polymerase (PDB 4MZ4). The compound is further anchored by hydrogen bonds between the 2-pyridone moiety and the backbone carbonyl atom of Gln446 and the NH group of Tyr448. The carboxylate and the quinoline N1-atom are linked to different residues via water-mediated hydrogen bond networks (the second sphere of water molecules and beyond was omitted for clarity). A second conformation of the Cys366 was omitted as well.

In summary, the discussed reports point out the high potential of SNAr chemistry in TCI discovery. Although only few medicinal chemistry programs have been published and little systematic investigation has been performed, the favorable properties of SNAr warheads such as tunability, synthetic accessibility, and structural rigidity will probably stimulate further exploration of this structure class. Moreover, electron-deficient (hetero)arenes are already present in many known ligands and approved drugs with excellent ADME properties. If a properly positioned nucleophile is present in the binding site, such a ligand might be readily turned into an irreversible modifier simply by the installation of suitable leaving groups. Besides the efforts discussed above, it is worth mentioning that an elegant approach to target DNA cytosine-5-methyltransferases covalently via an SNAr mechanism has very recently been reported in two studies by Akira Matsuda, Satoshi Ichikawa, and co-workers.(140,141) However, their approach addressing an active site cysteine via a suicide mechanism is beyond the scope of this Perspective. The interested reader is therefore referred to the original articles.

2.7. Cysteine Alkylation by Strain Release Reagents

A very elegant method for selective cysteine labeling by using strain release reagents was described in 2016 by the group of Phil Baran.(142,143) Although the strain-promoted addition of thiols to [1,1,1]-propellane derivatives has been known since the 1980s,(144) this reaction has not been applied to cysteine conjugation until recently. While [1,1,1]-propellane was found to be a useful building block for the derivatization (“propellerization”) of alkylamines by means of “turbo-Grignard” reagents,(145) phenylsulfonyl bicyclobutanes (e.g., compound 51, Figure 18) proved to be suitable for cysteine labeling applications. These bench-stable reagents selectively cyclobutylated cysteine in glutathione and in highly functionalized peptides under basic conditions while leaving other nucleophilic amino acid side chains untouched (Figure 18).(142) Analogous cyclopentylation reactions were carried out even in a stereospecific manner by using chiral 1-(phenylsulfonyl)bicyclo[2.1.0]pentanes (housanes, e.g., compound (+)-52) as the strain-release reagent.(143) The reactivity of the strained cycles could be readily tuned by modulating the electronic properties of the aryl system linked to the sulfonyl group. As expected, the attachment of electron-withdrawing substituents increased reaction rates while electron-donating substituents had the opposite effect. The adducts were chemically stable even in the presence of TCEP, but stability studies in a more physiologically relevant setting remain elusive. In a GSH reactivity assay, previously reported by researchers from Pfizer (vide infra),(12) strain-release warheads were benchmarked against common α,β-unsaturated amides and vinyl sulfonamides (see Table 1 for a representative selection). Half-lives spanned from 4 h for the 3,5-difluoro derivative (no analogues with stronger electron-withdrawing groups have been evaluated) to 19 h for the 4-methoxy analogue. The compounds thus proved to be significantly less reactive than the common N-phenylacrylamide motif (t1/2 = 0.9 h) or N-(vinylsulfonyl)pyrrolidine (t1/2 = 0.53 h) but in the same range as N-benzyl acrylamide (t1/2 = 15 h).

Figure 18

Figure 18. Strain-release reagents and their reaction with a cysteine-containing peptide.

Table 1. Influence of the Aryl Substituent on the Reactivity of Phenylsulfonyl Bicyclobutanes in a GSH Stability Assay and Comparison with Common Acrylamides
Due to their well-balanced and tunable reactivity toward cysteines along with the limited spatial requirements and the defined geometry, these electrophiles might be well-suited as warheads in drug discovery. Moreover, the replacement of the aryl sulfone moiety by analogous sulfonamides, carboxamides, esters, or ketones or the attachment of substituents to the strained ring system may enable the modulation of reactivity over a wider range. Unfortunately, no studies demonstrating the applicability of strain-release warheads in a complex biological setting have been published so far. One potential issue with this CRG is the underlying synthetic chemistry. Although Baran and co-workers have developed an operationally simple protocol for the preparation of the strain-release reagents discussed above, the flexible implementation of the latter into chemically more complex settings may require optimization and profound chemical expertise.

2.8. Cysteine Alkylation by Nucleophilic Displacement of Alkyl Halides

Although aryl halides are abundant in approved and investigational drugs, their alkyl counterparts are rare, with the notable exception of DNA alkylating agents(146) and unreactive alkyl fluoride motifs, that have been frequently applied in drug discovery, e.g., as bioisosteres or for modulating physicochemical properties.(147,148) The most representative sp3 halides used to covalently modify biological targets are α-halomethylketones and the analogous esters and amides. α-Halomethylketones have a long history as protease probes where the keto group is attacked by the active site nucleophile while the proximal alkyl halide irreversibly labels the enzyme, for example, by reacting with a histidine of the catalytic triad.(7) Mometasone, an approved corticosteroid, also contains an α-chloromethylketone residue. Fluoroalkyl groups can be found as the reactive moieties in mechanism-based inhibitors such as eflornithine(149) or trifluoromethyl deoxyuridine derivatives.(150) Moreover, fluoromethylketone (FMK)-tagged adenine analogues were among the first rationally designed covalent kinase inhibitors.(151,152)
The nucleophilic substitution reactions discussed here proceed via an SN2-mechanism (Figure 19A), in which the reaction rate depends on the nucleophile, the substrate, and the leaving group. SN1 reactions, where the rate depends solely on the loss of the nucleofuge to generate a highly reactive carbocation, are not utile in the context of TCI design. Nucleophilic substitutions of nonactivated linear haloalkanes are relatively slow, and increasing steric bulk around the electrophilic carbon center further decreases reactivity. Benzyl or allyl halides are more reactive because the transition state of the SN2-reaction (but also the carbocation in the case of an SN1-mechanism) is stabilized via conjugation with the π-system. A tremendous increase in reactivity is typically observed for α-halomethylketones, which may be rationalized by a further stabilization of the SN2-transition state via conjugation, or by the dual attraction model (Figure 19B).(153) However, the relative increase in reaction rates depends on the nucleophile and is typically more pronounced when strong/negatively charged nucleophiles are employed.(154) Nucleophilic substitution of α-halomethylcarbonyl compounds exclusively proceeds via an SN2-mechanism even if good leaving groups are employed, which can be attributed to the electron-withdrawing carbonyl group destabilizing the intermediate carbocation in SN1 reactions. Reactivity of α-halomethylcarbonyl compounds decreases if +M substituents are attached to the carbonyl group. Consequently, α-halogenated esters are less reactive than the corresponding ketones and the respective amides possess an even lower reactivity.(153) On the other hand, reactivity rises with increasing leaving group properties of the halogen (I > Br > Cl > F). α-Haloacetamides are quite versatile and can be used to label various nucleophiles (typically Cys but also Lys, His, Tyr, activated Ser, and Thr etc.). Iodoacetamides, for example, are highly reactive and known as cysteine capping reagents.(155) Iodoacetamide-derived clickable probes have recently been used to identify hyper-reactive cysteines by means of chemical proteomics.(156) In contrast, α-bromoacetamides, which feature a slightly lower reactivity,(12) are far less common. α-Chloroacetamides, which are the most frequently employed haloacetamides in covalent ligand design, possess further reduced reactivities. Their stability against GSH at pH 7.4 is in the same range as the one of α,β-unsaturated amides,(12) and a linear chloroacetamide-derived alkyne probe showed only moderate levels of labeling in soluble mouse liver proteomes.(157) In the case of aniline-derived α-haloacetamides, GSH reaction rates correlate with the Hammett parameter of the aryl substituent, i.e., electron-donating substituents decrease reactivity and vice versa.(158) Reactivity of α-haloacetamides can be further decreased by adding steric bulk in proximity to the reaction site. Therefore, the reaction rates of α-bromopropionamides are lower than the ones of α-chloroacetamides and α-chloropropionamides are even significantly less reactive than for example N-benzyl acrylamide (Figure 19C).(12) In support of the low and specific reactivity of α-chloropropionamides, a recent ABPP-based study identified (S)-CW3554 (53, Figure 19D) as an irreversible inhibitor of the protein disulfide isomerase A1 (PDIA1) with good selectivity in HEK293 cells.(159) Interestingly, the analogous (R)-α-chloropropionamide specifically labeled a distinct protein, the aldehyde dehydrogenase ALDH2, highlighting the role of the stereochemistry at the α-position of the warhead. However, it should be noted that both enzymes contain strongly nucleophilic active site cysteines and the potential of α-chloropropionamides in TCI design remains to be demonstrated.(160,161)

Figure 19

Figure 19. Alkyl halides as CRGs (A) General mechanism of the SN2 reaction. (B) Dual attraction model rationalizing the enhanced reactivity of α-halocarbonyl compounds. (C) Reactivities of α-halopropion- and acetamides in a GSH assay. Half-lives were determined in the presence of 10 mM GSH at pH 7.4 and 37 °Ca or 60 °Cb. N-Phenylacrylamide is shown for comparison. (D) 2-Chloropropionamide (S)-53, a covalent PDIA1 inhibitor.

As an example of cysteine targeted alkyl halides devoid of an α-carbonyl group, 1,4-disubstituted 5-chloromethyl-1,2,3-triazoles have recently been identified by Alexander Adibekian and co-workers as inhibitors of the O6-methylguanine-DNA-methyltransferase (MGMT), a noncatalytic DNA repair protein that possesses an active site cysteine.(162) The approach was inspired by N-heterocyclic carbene ligands, in which the reactive metal center is shielded by bulky substituents. Analogously, it was hypothesized that the reactivity of the 5-chloromethyl substituent can be modulated sterically by substituents at the N1- and the C4-position of the triazole ring. Ideally, these substituents would at the same time confer selectivity for a given target. A promiscuous clickable probe (54) was first synthesized and reacted with the proteome of MCF7 cancer cell lysates. MGMT was identified as one among the multiple labeled proteins. A small library of triazoles was synthesized by uncatalyzed or ruthenium-promoted azide–alkyne cycloaddition(163) and analyzed in a competition assay against probe 54. In this screening, analogue AA-CW236 (55) was identified as a highly potent MGMT inhibitor (KI = 24 nM), albeit with slow inactivation kinetics (kinact = 0.03 min–1) (Figure 20). The compound did neither cross-react with any of the targets of probe 54 at a concentration of 300 nM nor label the MGMT C145A mutant. Furthermore, MS-based proteomics indicated a clean selectivity profile. The compound, in combination with the DNA alkylation agent temozolomide, significantly increased the O6-alkylguanine levels in MCF7 cells compared to temozolomide alone, indicating a sensitization to the chemotherapeutic drug. Because of the low inactivation rates, however, it is not unlikely that the bulk of the observed effects are promoted by the potent reversible interaction.

Figure 20

Figure 20. 5-Chloromethyl-1,2,3-triazoles as covalent MGMT inhibitors.

Overall, alkyl halides, especially α-haloacetamides, seem well suited as warheads for noncatalytic cysteine residues. Key to the modulation of their reactivity is the nature of the leaving group and the steric environment, especially the substituent at the α-position, which needs to be tolerated by the binding site. Replacing halogens with alternative and tunable leaving groups such as (activated) esters or sulfonates may offer further options to achieve optimally balanced reactivity.

2.9. Cysteine Alkylation by Epoxides and Other Three-Membered Heterocycles

Epoxides (oxiranes) have a long history as warheads to target different types of proteases(7,164) and glycosidases.(165,166) In contrast, other three-membered heterocycles such as aziridines(167) and thiiranes(168,169) have received less attention. The reactivity of these heterocycles arises from their ring strain. Epoxides react with nucleophiles via SN2 mechanisms and in the absence of an acid catalyst, nucleophilic attack preferably occurs at the sterically less hindered position. Epoxides are used as CRGs in approved drugs, e.g., the epoxyketone-based proteasome inhibitor carfilzomib (56, Figure 21)(170) or the antibiotic fosfomycin (57).(171) Fosfomycin is a covalent modifier of bacterial UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) targeting a nonessential cysteine (Cys115 in E. coli).(172,173) Despite being structurally simple, this orally available drug can be safely administered in multigram doses, illustrating the tolerability of epoxide-derived compounds.(171) Moreover, epoxides can behave unreactive even if a proximal cysteine is present in the target’s binding site (e.g., trapoxin A in complex with HDAC 8(174)) and some approved drugs, e.g., ixabepilone (58),(175) feature a bystander epoxide, suggesting that this structural motif cannot be considered a safety issue per se. Styrene oxide has recently been shown to possess a similarly low reactivity as acrylamide or ampicillin toward GSH.(131) On a proteomic scale, terminal alkyl and spiro-epoxides have shown little or no protein labeling.(157) However, despite these facts, epoxides are often regarded with skepticism by medicinal chemists, which probably traces back to the well-known toxic effects of certain epoxy-metabolites as well as their DNA alkylating potential.(6) Despite the wealth of published literature, systematic studies on the factors determining the reactivity of epoxides and other three-membered heterocycles in a complex biological environment remain sparse. Another reason for the low abundance of epoxides in TCI discovery campaigns is their facile hydrolysis by epoxide hydrolases, and factors determining metabolic stability of this compound class are not fully understood. On the other hand, this liability might be used to confer kinetic selectivity in a similar manner as the fumaric acid esters discussed in section 2.1 do. Moreover, the small size and the possibility of being attacked at two proximal positions make epoxides quite versatile CRGs. Two examples for the application of epoxides in the rational targeting of noncatalytic cysteine residues are highlighted below.

Figure 21

Figure 21. Examples of epoxide-containing drugs.

A publication from 2010 by Alessio Lodola and co-workers describes a series of α-acyl epoxides as inhibitors of wild-type EGFR.(176) Notably, other electrophiles such as activated phenyl esters, carbamates, nitriles, and heterocycles containing an electrophilic sulfur atom were also evaluated in this study. Starting from the covalent inhibitor PD168393 (59, Figure 22), replacement of the acrylamide moiety by three different epoxide-containing residues furnished highly potent compounds (IC50 = 0.5, 0.5, and 1.2 nM for 60a, 60b, and 61, respectively) with cellular activities in the low nanomolar range. In contrast, the β-naphthylamine-derived control compound 62 did not show any activity in the A431 cell assay, suggesting the absence of general cytotoxicity. In an LC-MS-based GSH binding assay, epoxide 60a did not show any adduct formation after 1 h, while a 36% conversion was found for acrylamide 59. Irreversible binding of the epoxides was suggested by washout experiments, however, no further analysis of binding kinetics was performed and no confirmation of covalent target modification by MS or X-ray crystallography reported. Therefore, it cannot unambiguously be concluded if covalent binding took place. Notably, Daniel Rauh and co-workers observed only very weak activity of the analogous epoxide 63 on the cSRC S345C mutant sharing an equivalently positioned cysteine.(177)

Figure 22

Figure 22. α-Acyl epoxides as warheads for putatively covalent EGFR inhibitors.

In a similar approach from our own group, an epoxide moiety was used to invert the isoform selectivity profile of ruxolitinib (64, Figure 23), an approved JAK1/JAK2/tyrosine kinase (TYK) 2 inhibitor.(178) In search of selective JAK3 inhibitors, molecular modeling proposed that replacing ruxolitinib’s (R)-3-cyclopentyl propanenitrile side chain by a propylene oxide moiety could address JAK3 Cys909, which is unique within the JAK family. We prepared a series of ruxolitinib-derived triazoles and epoxy analogue 65 appeared to be a potent JAK3 inhibitor (IC50 = 35 nM) with a high selectivity (70–160-fold) in the JAK family. Because the other JAK family members possess a serine at the position equivalent to Cys909, the selectivity shift, which was not observed for nonreactive control compounds, suggests a covalent interaction with Cys909. However, covalent modification was not unambiguously proven since we discontinued this series in favor of tricyclic covalent-reversible JAK3 inhibitors.(179)

Figure 23

Figure 23. Ruxolitinib-derived triazoles with a propylene oxide warhead as selective JAK3 inhibitors.

Aziridines have been used for targeting active site carboxylates in glycosidases,(180) but reports on their application as CRGs in chemical biology and drug discovery remain sparse. Their avoidance can probably be traced back to the well-known DNA-alkylating properties of aziridinium ions, which are famous as the active species generated from N-lost derived chemotherapeutics.(181) Nevertheless, a recent example employing an aziridine as TCI warhead was provided by the groups of Kevan Shokat and John Burke.(182) With the aim of covalently addressing the oncogenic K-Ras G12D mutant via the aspartate, different electrophilic head groups were attached to an optimized irreversible ligand of K-Ras G12C (general structure 66, Figure 24). Despite the reactivity of the representative aziridine 66a toward carboxylate groups in solution, negligible modification of K-Ras G12D was observed. In contrast, the oncogenic K-Ras G12C mutant was fully labeled. Binding was independently confirmed by differential scanning calorimetry and by hydrogen–deuterium exchange MS (HDX-MS). X-ray-crystallography and MS finally revealed the exact binding mode (Figure 25). It is worth mentioning that the attack of the thiol group occurred at the sterically more hindered α-carbon atom.

Figure 24

Figure 24. K-Ras G12D or G12C-targeted covalent inhibitors. Key compound 66a features an aziridine warhead.

Figure 25

Figure 25. X-ray crystal structure of the K-Ras G12C mutant covalently bound to compound 66a (PDB 5V6V). Cys12 forms the covalent bond by opening the aziridine ring at the β-position. The indole NH and quinazoline N1-atom are involved in charge-assisted hydrogen bonds to the side chains of of Asp69 and Arg68, respectively, while the piperidine carboxamide oxygen interacts with the side chains of Tyr96 and Asp92 via water-bridged hydrogen bonds.

2.10. Cysteine Targeting by Nitroalkyl Groups As Masked Electrophiles

The covalent complex formation between 3-nitropropionate (3-NP, 67a, Figure 26) and Mycobacterium tuberculosis isocitrate lyase (ICL) was recently investigated by the group of Andrew Murkin. They found this compound to irreversibly inhibit ICL without the requirement for cofactors or prior redox-activation of the nitro group.(183) After 20 h of incubation with 3-NP, only negligible enzyme activity could be recovered by jump dilution, an observation that was not influenced by the addition of DTT. However, the presence of glyoxylate increased reaction rates, suggesting cooperative binding. ESI-MS and X-ray crystallography (PDB 6C4A) revealed the formation of a thiohydroximate by reaction of the inhibitor with Cys191, an active site residue acting as a general base in ICL catalysis.(184) An inverse solvent isotope effect indicated the thiolate form to be the nucleophilic species. The reaction could be rationalized by the CH-acidic nature of 3-NP (pKa = 9.0) generating significant amounts of the conjugate base propionate-3-nitronate (P3N, 67b) at neutral pH. A mechanism was proposed in which Glu285 protonates P3N at an oxygen atom to transform the nucleophilic nitronate into an electrophilic nitronic acid. The latter could react with Cys191 to form the thiohydroximate by water elimination, probably via a second protonation step involving Arg228. The reaction mechanism was supported by kinetic analyses showing that preformed P3N (kinact/KI = 2.6 × 104 M–1 s–1) inactivated the enzyme approximately 100 times faster than 3-NP. Accordingly, nitroalkanes should be suitable as cysteine-targeted warheads for proteins hosting a proper combination of a sufficiently reactive cysteine and proximal acidic residues capable of generating the nitronic acid electrophile. Besides the aforementioned concerns on nitro groups in drug discovery, the generalizability of this concept remains to be demonstrated. To this end, chemoproteomic studies with nitroalkyl-derived probes would provide valuable information on the scope of this reactivity with respect to other target proteins.

Figure 26

Figure 26. 3-Nitropropionate, propionate-3-nitronate, and the suggested reaction mechanism with ICL.

2.11. Reversible Cysteine Addition to α-Cyanoacrylamides

Covalent reversible targeting of noncatalytic cysteines has recently emerged as a strategy to benefit from the advantages of irreversible TCIs (e.g., long residence times and the (potentially) increased selectivity) while avoiding liabilities like permanent off-target modification and the risk of idiosyncratic toxicity.(28) The rational implementation of this concept in TCI design has been pioneered by the group of Jack Taunton, who introduced the β-substituted α-cyanoacrylamide functionality as a promising covalent-reversible warhead for cysteine.(25) Although this electrophile can be considered highly reactive toward thiols, the increased α-CH acidity and thermodynamic destabilization of the β-thioether adduct(185) favor the reverse reaction and thereby dissociation from off-targets or peptides, which are not capable of stabilizing the covalent complex by noncovalent interactions. Interestingly, a recent crystal structure of the kinase JAK3 in complex with the selective α-cyanoacrylamide-based inhibitor FM-409 (68, Figure 27) shows the coexistence of both the covalently bound and the unreacted form (PDB 5LWN), supporting the notion of covalent-reversible binding.(179) By adding steric bulk to the β-position of the α-cyanoacrylamide(11) or by replacing the amide group by an electron-withdrawing heteroarene,(186) the intrinsic reactivity of this CRG and the dissociation rates of the derived covalent-reversible inhibitors can be modulated.(185) More details are provided in a recent Perspective article(40) and subsequent publications highlighting the merit of this warhead class in the design of EGFR-(187) and highly isoform-specific JAK3 inhibitors.(188) Global profiling of the covalent interactions of this chemotype in proteomes, however, is complicated by the reversible nature of protein modification and only a few studies assessing target and off-target binding in cells have been published to date. For example, compound 69 (Figure 27), a low micromolar covalent-reversible inhibitor of glycosaminoglycan (GAG) sulfotransferases(189) was tested for competition with a known alkynylated iodoacetamide probe.(13,156) The compound neither competed with iodoacetamide-labeling in some isolated proteins containing hyper-reactive cysteines(156) nor did it change the labeling profile in the cellular proteome of Neu7 astrocytes, indicating good specificity despite the highly reactive warhead.(189) The compound further showed good stability in rodent and human liver microsomes and moderate in vivo PK properties after intravenous injection in rats. In another study, prolonged cellular target engagement has been demonstrated for α-cyanoacrylamide-derived covalent-reversible BTK inhibitors (e.g., compound 70, Figure 27) in isolated Ramos B cells and in PBMCs after oral dosing in rodents.(11) Notably, further development of this compounds furnished PRN1008,(190−192) a covalent-reversible oral BTK inhibitor of undisclosed structure currently being in phase II/III clinical trials for the treatment of pemphigus (NCT02704429/NCT03762265 on https://www.clinicaltrials.gov) and immune thrombocytopenic purpura (NCT03395210). A dually activated Michael acceptor is also present in the approved catecholamine-O-methyltransferase (COMT) inhibitor entacapone (71).(193) Although further studies are clearly required to determine the biological fate of α-cyanoacrylamides and analogous Michael acceptors, the currently available data suggest them to be promising warheads for in vivo use.

Figure 27

Figure 27. α-Cyanoacrylamide-derived covalent-reversible inhibitors.

Beyond α-cyanoacrylamides, benzylidene rhodanines have been reported as covalent-reversible Michael-type warheads.(136) However, covalent binding does not seem to be a general feature of this compound class.(194) In contrast, arylidene dinitriles are very reactive reversible Michael acceptors, but the reverse reaction seems to be competed by side reactions in this case.(188) Moreover alkynone-derived Michael acceptors (cf. section 2.3)(79) and β-thiomethyl α-cyanovinylsulfones(195) have recently been shown to react reversibly with thiols. According to the mechanism in Figure 5B, however, the reverse reaction is rather a thiol exchange, which might complicate the implementation in TCI design. Nevertheless, the current combinations of electron-withdrawing groups for generating covalent-reversible Michael acceptors with specifically tuned reactivities are by far not exhaustive, leaving room for further optimization. It will be interesting to see if alternative chemotypes such as vinyl sulfones or sulfonamides equipped with an additional electron-withdrawing group in the α-position can further extend the scope (e.g., toward lysines, vide infra) of this covalent-reversible chemistry.

2.12. Reversible Cysteine Addition to Aldehydes

Aldehydes and ketones are common warheads in proteolytic enzyme inhibitors.(7,26,196) They react reversibly by forming tetrahedral hemi(thio)acetals and ketals mimicking the transition state of amide bond cleavage.(197) These moieties, however, are far less commonly employed to address noncatalytic cysteine or lysine residues (some recent examples for lysine targeting by Schiff base formation can be found in refs (198−200)). In a recent drug discovery program by Fairhurst and co-workers, the utility of aldehydes in covalent-reversible kinase targeting has been demonstrated.(132,201) In the screening campaign, which also identified SNAr-based irreversible inhibitor 41 (see section 2.6), a set of closely related 2-formylquinoline amides exemplified by compound 72 (Figure 28, IC50 = 65 nM) was discovered. These compounds were potent FGFR4 inhibitors with good selectivity in the FGFR family but also against the FGFR4 C552A mutant and a panel of over 50 kinases. Replacement of the formyl warhead by a proton, hydroxymethyl, or acid group was detrimental to activity. In accordance with these data, modeling studies suggested covalent-reversible binding to the middle-hinge Cys552 by hemithioacetal formation. The quinoline nitrogen atom and the carboxamide proton were found to be of crucial importance because they stabilize a pseudotricyclic arrangement via intramolecular hydrogen bonding, thereby positioning the aldehyde for covalent interaction. Moreover, the electron-withdrawing ortho-quinoline scaffold further increases the aldehyde’s electrophilicity. Although certain questions concerning toxicity and metabolic stability of the aldehyde moiety remained,(202) this compound class was selected for optimization due to its excellent potency and selectivity profile. Covalent-reversible inhibition was considered more promising than irreversible targeting due to the short resynthesis half-life (<2 h in HUH7 and Hep3B cells) of FGFR4. The 2-formylquinoline amide scaffold, however, was unsuited for further development due to very low solubility. A scaffold morphing approach increasing the sp3 portion led to bioisosteric 2-formylpyridine ureas (exemplified by 73ad) with improved solubility. Replacement of the trifluoromethyl (R1) group by a nitrile (73b) decreased lipophilicity, and the introduction of an oxygen atom in the saturated ring system (73c) or polar R2 substituents (73d) further increased both solubility and potency. The advanced lead compound 73d featured low nanomolar potency in an enzymatic and a Ba/F3 cell assay (IC50 = 1.3 and 18 nM, respectively). Residence times (pH 7.0) for the slightly less potent compounds 73ac were in the range between 105 min (73c) and 272 min (73a) and thus much increased compared to an equipotent noncovalent inhibitor (<1.4 min). The significant off-rates (between 3.7 × 10–3 and 9.6 × 10–3 min–1) pinpoint the highly reversible nature of hemithioacetal formation.(203) Consistently, no covalent adducts were found in mass-spectrometric experiments. Although no inhibitor-bound X-ray crystal structure was determined, SAR and modeling provided insight into the binding interactions. Replacement of the pyridyl side chain by pyrimidine and pyrazine was tolerated with a slight loss in potency, while the use of five-membered N-heterocycles was detrimental to activity. Methylation of the urea NH furnished a completely inactive compound. Constraining and opening the piperidine ring was also tolerated, while increasing the size of this saturated ring system by one methylene group led to a substantial potency loss.

Figure 28

Figure 28. Optimization of covalent-reversible FGFR4 inhibitors possessing an aldehyde warhead.

The advanced lead compound 73d was further optimized to furnish clinical candidate FGF401 (74), a compound with a good oral bioavailability, PK, and safety profile, which is currently in phase I/II clinical studies (NCT02325739) for FGFR4 and β-klotho positive solid tumors and hepatocellular carcinoma.(204) Although the final optimization steps as well as the outcome of clinical evaluation remain yet to be reported, the presented preclinical investigations emphasize that aldehydes, despite potential metabolic liabilities, can indeed be valuable warheads for the covalent-reversible targeting of cysteines beyond the active sites of hydrolases.

2.13. Reversible Cysteine Addition to Activated Nitriles

Besides aldehydes, nitriles have a long history as covalent-reversible warheads for protease inhibitors(196,205) and are employed in approved drugs such as saxagliptin.(206) As indicated by their prevalence in noncovalent inhibitors,(207) nitriles behave relatively inert, and covalent adduct formation generally requires highly reactive active site nucleophiles along with precise positioning of the electrophilic carbon atom. However, the electrophilicity of the nitrile group can be increased by the attachment to electron-withdrawing moieties, e.g., heteroaryl rings, alkylamines,(12,208) or acylated N,N′-dialkylhydrazines.(209)
Pyridine and pyrimidinecarbonitriles show tunable reactivities toward glutathione, which are in a similar range as the ones of acrylamides (Figure 29A,B), suggesting these structural elements as suitable CRGs for targeting noncatalytic cysteines. Nitriles reversibly react with cysteines to form thioimidates via a Pinner-type addition, as it has been shown by X-ray crystallography for the cysteine proteases cathepsin S (PDB 3N3G)(210) and the adenovirus protease adenain (PDB 4PIQ).(211) In the case of 2-pyridinecarbonitrile analogues, a double activation mechanism involving the pyridine nitrogen atom as a general base to deprotonate the cysteine while concurrently activating the nitrile group has been proposed.(210) A recent study aiming to capitalize on the increased reactivity of 2-pyridinecarbonitriles compared to their phenyl counterparts was published by Pamela England and co-workers.(212) They hypothesized that a nitrile group present in the approved nonsteroidal antiandrogen bicalutamide (compound 75, Figure 30A) could be activated to react with the proximal Cys784 in the androgen receptor (AR), a residue that is not among the over 160 point mutations, which have been associated with prostate cancer. DFT calculations predicted a 10-fold increase in electrophilicity by introduction of a pyridine nitrogen atom ortho to the bicalutamide nitrile group (compound 76a). Reactivity studies with 76a and free cysteine in pH 7.4 buffer showed a 99% conversion to the corresponding thiazoline within 4 h, while no S-adduct formation was observed for 75 after 24 h. The additional nitrogen atom increased binding affinity approximately 150-fold (KI = 0.15 nM) compared to the parent compound bicalutamide. In a cellular luciferase reporter assay detecting AR-mediated transcription, a substantial increase in activity (IC50 = 0.015 vs 0.31 μM for 76a and 75) was confirmed, while the two analogues 76b and 76c, devoid of the nitrile group, were much weaker inhibitors (IC50 = 1.41 and 1.33 μM, respectively). A mechanism was proposed in which Arg752 activates the nitrile group via hydrogen bonding (Figure 30B). Although not explicitly mentioned, further activation could result from a hydrogen bond between the pyridine nitrogen atom and the cysteine’s thiol, as mentioned above,(210) to facilitate the nucleophilic attack. NMR studies to confirm the covalent-reversible binding, however, were hampered by protein precipitation, and mutation of Cys784 prevented proper folding. Furthermore, no structural data was reported and an ultimate proof of the suggested covalent interaction remains elusive.

Figure 29

Figure 29. Reactivities of activated nitriles in a GSH-based assay. Half-lives (increasing from left to right) were determined in the presence of 10 mM GSH at pH 7.4 and 37 °C. Data for common acrylamides are provided for comparison.

Figure 30

Figure 30. Bicalutamide-derived antiandrogens with a putative covalent-reversible binding mode. (A) Chemical structures of the compounds studied by England and co-workers. (B) Suggested mechanism of covalent binding to Cys784 of the androgen receptor.

Cyanamides show similar reaction kinetics with GSH as acrylamides (see Figure 29) and should therefore be applicable in nonactive site cysteine targeting. The reversible thiol addition furnishes isothioureas, as it has been confirmed for cathepsin K by X-ray crystallography (PDB 1YK7).(213) While cyanamides are not uncommon in cysteine protease inhibitors,(213−216) reports on targeting noncatalytic cysteines using this functionality are rare. When designing cyanamide-based warheads, it should be kept in mind that free protons at the amino group could, in principle, enable the tautomerization to carbodiimides,(217) which have distinct reactivities. It remains to be tested if this equilibrium is relevant under physiological conditions.
Micah Benson and colleagues from Pfizer reported the cyanamide-based inhibitor PF-303 (77, Figure 31), which was used as a chemical in vivo probe to investigate the phenotype of BTK inhibition in mice.(218) The underlying medicinal chemistry program was not published, but PF-303 was claimed to be highly potent (IC50 = 0.64 nM) and orally bioavailable. The compound, which can be considered as a pseudorigid cyanamide analogue of the covalent BTK inhibitor ibrutinib, possessed a kinact/KI ratio of 1.44 × 105 M–1 s–1, which is in the same range as for many acrylamide-derived kinase inhibitors.(21,219) Binding was reversible with a dissociation half-life of 5 h. The compound also inhibited the kinases BMX and TEC featuring an equivalently positioned cysteine. In contrast, an over 10000-fold selectivity against JAK3 and ITK also sharing this cysteine was observed. The latter finding is in line with SAR reported for a series of ibrutinib-derived ITK inhibitors, where replacement of the acrylamide moiety by cyanamide completely eroded the inhibitors’ activity.(220) PF-303 showed high selectivity against the related Src family kinases and potently inhibited anti-IgM F(ab′)2-promoted B-cell proliferation with an IC50 of 2 nM.

Figure 31

Figure 31. PF-303, a covalent-reversible BTK inhibitor featuring a cyanamide warhead.

2.14. Reversible Cysteine Addition to Isothiocyanates

Isothiocyanates have long been known as natural electrophilic compounds in foods, and their presence has been associated with health benefits. They mainly occur in cruciferous vegetables as metabolic breakdown products of glucosinolates.(221) As a representative of this compound class, allyl isothiocyanate (78, Figure 32A) causes the pungent taste of mustard and wasabi, which has been attributed to TRPA1 channel activation by covalent cysteine (and lysine) binding.(222) Various clinical trials in all phases have been conducted with phenethyl isothiocyanate (PEITC, 79)(221) and sulforaphane (80), e.g., for cancer chemoprevention and the treatment of schizophrenia and autism spectrum disorders (see https://clinicaltrials.gov). Evaluation in a clinical setting and the natural abundance in foods suggest that these electrophiles are well tolerated, however, their biological action has been attributed to multitarget effects. Isothiocyanates rapidly undergo GSH addition to form dithiocarbamates, but this process is reversible(223,224) and trans-thiocarbamoylation to proteins occurs.(225) Reactivity toward amino groups to form stable thioureas is much lower. Nevertheless, lysine residues can be labeled as a consequence of the dithiocarbamate–isothiocyanate equilibrium,(226) but also direct reaction pathways seem possible (Figure 32B). Reactivity of isothiocyanates can be expected to increase with the attachment of electron-withdrawing groups, while electron-donating (+I) substituents should decrease the electrophilicity of the sp-hybridized carbon center.(224) Many proteins, such as glutathione S-transferase π (GSTP1),(227) the cysteine-rich protein Keap1,(228) protein tyrosine phosphatases,(229) or the protein kinase MEKK1,(230) are covalently labeled by isothiocyanates at cysteine residues. Binding to cysteines of tubulin has been suggested as a key mechanism of action of natural isothiocyanates,(231) and the N-terminal catalytic proline in macrophage migration inhibitory factor (MIF) is also modified by these compounds.(232) Because of the reversible nature of cysteine binding, investigation of targets in living systems is challenging. Despite of the wealth of data on isothiocyanates that has accumulated during several decades, systematic studies of SAR and applications in TCI design remain sparse. This might, at least in part, be attributed to the very complex equilibria driving isothiocyanate distribution and availability in vivo.(233)

Figure 32

Figure 32. Isothiocyanates as covalent-reversible warheads for cysteine and irreversible CRGs for lysine. (A) Common isothiocyanates found in cruciferous vegetables. (B) Reversible reaction of isothiocyanates with GSH or cysteines in proteins and slow thiourea formation, e.g., with lysine. Possible direct reaction pathways are depicted as dashed arrows.

2.15. Reversible Cysteine Addition to Electron-Deficient Heteroarenes Forming Stable Meisenheimer Complexes

In a recent publication, Campbell McInnes and co-workers described fragment-like covalent-reversible polo-like kinase (PLK) 1 inhibitors based on a benzothiazole N-oxide scaffold.(234) This compound class was identified by a virtual screening campaign and the initial hit 81a (Figure 33) possessed a moderate potency in the low micromolar range (IC50 = 2.5 μM). Subsequent SAR studies identified the nitro group in the C7-position and N-oxide as crucial features for activity, while replacement of the C2-carboxamide moiety by a nitrile or a hydroxamate group was tolerated with a moderate loss in potency. Lipophilic −I substituents were favorable at the C5 position, with the more potent compound 81b (IC50 = 0.4 μM) featuring a trifluoromethyl thioether group. Because compound 81b possessed limited ATP competitivity and docking suggested close proximity between the Cys67 side chain and the C4-aryl carbon atom, covalent binding via formation of a Meisenheimer complex was considered as the potential mechanism of action. It is worth mentioning that Meisenheimer complex formation has already been described earlier as the mode of action of the glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol.(235) The stability of such negatively charged σ-complexes can be rationalized by the very electron-deficient nature of the (hetero)aryl system combined with the low mobility of the leaving group, preventing its departure. It can further be assumed that a positive electrostatic surface potential in the protein binding pocket would be beneficial to stabilize such complexes. By using n-butylamine and 1-butanethiolate as surrogate nucleophiles in NMR and UV–vis experiments, potency was roughly correlated with the propensity of the compounds to form Meisenheimer-type adducts. As expected, the reaction was reversible upon removal of excessive n-butylamine. Consequently, it can be assumed that such inhibitors would be subjected to rapid but reversible GSH addition in vivo. Moreover, inhibitor 81a was completely inactive (IC50 > 100 μM) against the PLK1 C67S mutant. Screened at 100 μM, 81a showed good selectivity in two small kinases panels, including three kinases with an equivalently positioned cysteine. No details on cellular permeability, metabolic stability, and binding kinetics were provided, and no ligand-bound X-ray crystal structure was solved to confirm covalent modification of Cys67. Although being highly interesting from a conceptual point of view, it remains to be seen to what extend moieties capable of forming stable Meisenheimer complexes, which typically contain one or several nitro groups to stabilize the negative charge, will enrich our current medicinal chemistry toolbox of covalent-reversible warheads.

Figure 33

Figure 33. Meisenheimer complex-forming electrophiles as putatively covalent-reversible PLK1 inhibitors. Mechanism and selected compounds.

2.16. Reversible Cysteine Targeting by Disulfide Tethering

The formation of disulfide bridges is a key feature of cysteine residues in biological systems. Accordingly, cysteines can be addressed by strained or unstrained disulfides via thiol–disulfide exchange, by different reagents possessing electrophilic sulfur atoms, or even with free thiols under oxidizing conditions (see Figure 34A for some examples). Interesting recent applications include disulfide tethering for identifying low affinity fragments,(182,236) thiol-mediated uptake approaches,(237) and glutathione-responsive prodrugs.(238) Disulfide tethering can be very useful in structural biology.(239) For example, covalent trapping of the β2 adrenergic receptor, equipped with an engineered cysteine (H93C), by the disulfide-based ligand FAUC50 (82, Figure 34B) enabled the first X-ray crystallographic structure determination of this GPCR in an agonist-bound state.(240) Although many medicinal chemistry campaigns, especially in academia, have exploited disulfide formation for covalent cysteine targeting (for some recent examples, see refs (241−243)), it remains questionable if this linkage strategy is suitable for targeted covalent inhibition in vivo due to the complexity of physiological thiol–disulfide equilibria and redox systems.(244,245) Therefore, no further discussion is provided.

Figure 34

Figure 34. Reversible cysteine targeting by disulfide bond formation. (A) Selected examples of headgroups known to generate disulfide bonds. (B) FAUC50, a covalent ligand which enabled crystallographic structure determination of the β2 adrenergic receptor.

3. Nucleophilic Targeting of Oxidized Cysteines with Carbon Acids

ARTICLE SECTIONS
Jump To

Cysteine oxidation is known as an important regulator of protein function.(57) Oxidation of the thiol group with reactive oxygen species (ROS) furnishes sulfenic acids, which can further react following a variety of reversible and irreversible reaction pathways.(246) Reversible S-sulfenylation is involved in the regulation of phosphatases, kinases, ion channels, and a plethora of other proteins.(247) For example, sulfenylation of Cys797 in the EGFR kinase domain increases kinase activity while rendering the target insensitive to modification by covalent EGFR inhibitors.(248) In contrast to thiols, sulfenic acids are only weak nucleophiles while holding a more pronounced electrophilic reactivity. The latter has been capitalized for sulfenic acid detection with nucleophilic carbon acids.(249) The most prominent sulfenic acid sensors are based on 5,5-dimethyl-1,3-cyclohexanedione (dimedone, 83, Figure 35) and similar cyclic 1,3-dicarbonyl scaffolds. Other electrophilic groups that have been shown to trap sulfenic acids include norbornenes(250) and strained cycloalkynes,(251) the latter having simultaneous electrophilic reactivity toward thiols.(247)

Figure 35

Figure 35. Carbon acids adressing cysteine sulfenic acids. (A) Model system used by the Carroll group to assess sulfenic acid labeling with C-nuclophiles. A possible alternative reaction pathway is highlighted by the dashed arrow. (B) Cysteine sulfenic acid formation by ROS and trapping with dimedone. (C) Reactivities of cyclic C-nucleophiles. (D,E) Reactivities of linear C-nucleophiles. (F) Tofacitinib, an approved JAK inhibitor shown to react with sulfenic acids. Reactivity is expressed by pseudo-first-order rate constants derived from an LC-MS assay using the model system shown in (A).

The most notable contributions in targeting oxidized cysteines have come from the group of Kate Carroll, who made extensive use of α-CH acidic β-dicarbonyls and similar compounds for sulfenic acid sensing. For example, they used redox-based probes to monitor oxidation of the phosphatases’ active site cysteine in living cells.(249) As the reaction rate of sulfenic acids with dimedone is relatively low, the Carroll group developed a convenient LC-MS-based assay for screening the reactivity of C-nucleophiles toward a dipeptide-derived sulfenic acid under aqueous conditions. The principle relies on the in situ generation of the sulfenic acid from a more stable cyclic sulfenamide (Figure 35A).(247) A large number of cyclic(247) and linear(252) C-nucleophiles were screened. For cyclic C-nucleophiles, an increase in reactivity could be achieved, for example, by annellating a benzene ring and replacing one of the ketones by a carboxamide or sulfonamide group. The most reactive cyclic C-nucleophiles outperformed the reactivity of dimedone more than 200-fold as indicated by their pseudo-first-order rate constants determined in the above-mentioned assay (see Figure 35C for selected examples). The reaction products were stable against reduction with DTT, GSH, and TCEP, and no cross-reactivity with protected cysteine, cystine, serine, lysine, or a sulfinic acid was observed. However, it should be mentioned at this point that recent evidence suggests that sulfenamides can react faster with dimedone analogues than sulfenic acids.(253) Therefore, the reactivities observed in the aforementioned assay may at least in part arise from direct reaction with the employed sulfenamide precursor. In a model study, selected compounds showed near-quantitative labeling of the C64S/C82S mutant of the glutathione peroxidase (GPX) 3 at a single oxidized cysteine residue. Linear C-nucleophiles could also be tuned, however, within a narrower range (Figure 35D,E). Good reactivities were obtained by attaching electron-withdrawing α-groups to methyl sulfones. Interestingly, the reaction products of the most reactive derivative 85 could be readily cleaved under reducing conditions, a finding that was confirmed in a GPX3-SOH model and in HeLa cell lysates. In contrast, nitro analogue 84 formed stable products withstanding reductive cleavage for several days. Intriguingly, the cyanoacetamide group of the approved reversible JAK inhibitor tofacitinib (86, Figure 35F) was also shown to react with cysteine sulfenic acids, raising the question about potential off-target modification.
In a subsequent study, the same group investigated the cysteine “sulfenylome” of RKO colon adenocarcinoma cells.(254) To this end, five of the C-nucleophiles from the above studies were equipped with alkyne handles (8791, Figure 36) to enable click-chemistry-based tagging and enrichment. By employing MS-proteomics, 1283 S-oxidation sites in 761 proteins were identified. Because sulfenamides, which can be formed from sulfenic acids, but also from (mixed) disulfides and amides, are quite abundant in biological systems, it seems likely that the observed oxidation sites account for a combination of the two former oxidized species.(253) Nevertheless, little overlapping was observed with respect to the labeling sites of different nucleophiles, suggesting significant discrimination even at this low level of structural complexity. Despite the relatively slow reaction kinetics, these data indicate that selective targeting of cysteine sulfenic acids and sulfenamides with more optimized compounds should in principle be possible. Further investigations, including the application of such molecules in vivo, are highly anticipated.

Figure 36

Figure 36. Alkyne-tagged C-nucleophilic probes for chemical proteomics studies and their reactivity expressed by second-order rate constants derived from the model system shown in Figure 35A.

4. Targeting the Lysine Side Chain

ARTICLE SECTIONS
Jump To

Significant effort has recently been put into developing selective warheads for lysine residues, and key studies up to early 2017 have been summarized in a recent mini-review.(39) Lysine, together with cysteine, has been the most common residue addressed in bioconjugation chemistry.(41) Perhaps the most prominent labeling reagents for the lysine ε-amino group are highly activated N-hydroxysuccinimide (NHS)-esters, which are generally considered too reactive for application in TCIs. Lysine offers certain distinct advantages over cysteine, most notably the much higher abundance (5.8% vs 1.9%).(59) It is typically found on protein surfaces, on interfaces mediating protein–protein interactions as well as in binding cavities. Lysine residues play a role in catalysis, e.g., by acting as a base or as a nucleophile.(255) Moreover, lysines in active sites can assist catalysis by positioning reactive residues or activating them via (charge-assisted) hydrogen bonds (e.g., in kinases). In such cases, lysine residues are indispensable, and resistance mutations, as they may occur for noncatalytic cysteines,(65) unlikely. However, lysine has been much less frequently considered as a residue to be addressed by TCIs. The latter fact can be attributed to the special challenges associated with the ε-amino group as a nucleophile, especially when compared to cysteine. At physiological pH, surface-exposed lysine (pKa ≈ 10.5)(256,257) is almost entirely protonated (99.9%), rendering the side chain poorly nucleophilic. However, the pKa largely depends on the chemical environment and buried lysines can undergo a pKa shift of up to 5 units, making them addressable by electrophiles.(257) As discussed before (see section 2.4 and section 2.13), the ligand itself may also contribute to pKa perturbation. Besides protonation, post-translational modifications (most notably acylation) can make the lysine side chain unsusceptible toward electrophilic reagents. Another special feature of lysine is the long and linear architecture of the side chain causing a high degree of conformational freedom. Whether this property is to be considered favorable or not cannot be generalized and depends on the respective context.
By employing a similar approach as previously used for assessing the reactivity of cysteines in native biological systems,(13,156) the Cravatt group set out to identify ligandable lysine residues by means of chemical proteomics.(258) A highly activated ester probe equipped with an alkyne handle (compound 92, Figure 37) was used to quantify the reactivity of lysine residues in proteomes. The study recovered over 9000 reactive lysines from different human cell lines, a much higher number than previously found for cysteine with a iodoacetamide probe, that roughly reflects the relative abundance of these amino acids.(13,156) Experiments at different probe concentrations and competition experiments with various reactive fragments (general structures 9395, Figure 37) identified (hyper)reactive lysine residues that have a high probability of being addressable by small molecules in a selective manner. A fact particularly worthy of note is that such lysines were identified mainly in proteins not found in the DrugBank, including many challenging targets such as transcription factors and scaffolding proteins. However, no conserved motifs could be associated with lysine hyper-reactivity, and further insights are required to deepen our understanding of lysine nucleophilicity and ligandability.

Figure 37

Figure 37. Probes and warheads used to indentify ligandable lysines by chemical proteomics.

Lysine side chains can react covalently with Michael acceptors, but aza-Michael additions with lysines have often been discovered by chance rather than implemented by design. For example, the natural product wortmannin (96, Figure 38) labels Lys833 in phosphoinositide 3-kinase γ (PI3Kγ) by aza-Michael addition and concomitant opening of the ligand’s furan ring (Figure 38 and PDB 1E7U).(259,260) More recently, the Cheeseman group serendipitously discovered that an adenosine-derived inhibitor designed to target a cysteine (Cys17) residue in the heat shock 70 kDa protein 1 (HSP72) was in fact bound to a lysine side chain (Lys56).(261) In general, most Michael acceptors are more reactive toward the “softer” cysteine thiols (vide infra), which can be problematic as targeting the lysine ε-amino group may require more activated acceptors. The latter are more likely to exhibit cysteine-mediated off-target reactivity. The aza-Michael reaction with lysine furnishes Mannich-type bases which can re-eliminate the (protonated) amino group to recover the reactive electrophile. Thus, adduct formation can in principle be considered reversible.(262) The extent of the reverse reaction, however, largely depends on the chemical nature of the reaction product (i.e., α-CH acidity of β-amino ketones vs esters or amides and the basicity of the amino group) and it remains to be investigated if it could become relevant under physiological conditions.

Figure 38

Figure 38. Reaction mechanism of wortmannin with Lys833 in PI3Kγ.

4.1. Reactivities of Michael Acceptors and Nitriles toward Lysine versus Cysteine

Adam Gilbert and colleagues from Pfizer have recently assessed the reactivities of common electrophiles including α,β-unsaturated amides, α,β-unsaturated sulfones, α,β-unsaturated sulfonamides, cyanamides, and nitriles toward N-α-acetyl lysine and compared these with the corresponding GSH reactivities.(263) In the case of N-α-acetyl lysine, the assay was conducted at pH 10.2 (≈ 20% of unprotonated amine) to mimic the pKa perturbation in binding pockets while reactions with GSH were performed at pH 7.4. Although it is clear that this model system does not fully reflect the real situation in binding sites, it provides a reasonable picture on reactivity trends toward thiol(ate) and amino nucleophiles. Remarkably, no reaction was observed for N-α-acetyl lysine at pH 7.4 even with the most reactive electrophiles, pointing out the poorly nucleophilic nature of the protonated amine and confirming that the carboxylate group also remains inert. As expected, this model system recapitulated the same general trends as previous GSH reactivity studies (for selected examples, see Figure 39).(263) Reactions of α,β-unsaturated amides proceeded 1.2–5 times faster with glutathione than with N-α-acetyl lysine. Similarly, the GSH addition to N-cyanopyrrolidine was more than twice as fast compared to N-α-acetyl lysine, while the reaction of GSH with 2-cyanopyrimidine was even nine times faster. Interestingly, an opposite tendency was observed for α,β-unsaturated sulfones and the corresponding sulfonamides. For example, α,β-unsaturated sulfonamides reacted 1.6–4-fold faster with N-α-acetyl lysine compared to GSH, while an even nine times faster reaction was measured for methyl E-prop-2-enyl sulfone. This difference may be rationalized with the Pearson HSAB theory,(54) suggesting that the strongly electron-withdrawing sulfonyl group makes the β-position less polarizable (i.e., harder), favoring the reaction with a harder nitrogen nucleophile rather than with the soft thiol(ate) group. Although α,β-unsaturated sulfones and sulfonamides have a higher general reactivity compared to the corresponding acrylamide-derived Michael acceptors, sterical and electronic tuning of these electrophiles might be harnessed to generate selective lysine-targeted probes with a low degree of promiscuity.

Figure 39

Figure 39. Reactivity of different Michael acceptors and activated nitriles toward lysine and GSH. Compounds with a preference for GSH are shown the upper row, while such preferably reacting with N-α-acetyl lysine are depicted in the lower row. Half-lives were determined in the presence of 50 mM N-α-acetyl lysine at pH 10.2 and 37 °C or 10 mM GSH at pH 7.4 and 37 °C.

The higher reactivity of α,β-unsaturated sulfones toward lysine was used by the groups of Bernard Golding and Jane Endicott to generate the first irreversible inhibitor of cyclin-dependent kinase (CDK) 2.(264) The key compound of this study, vinyl sulfone NU6300 (98a, Figure 40A), was identified while optimizing sulfonamide NU6102 (97). Binding of 98a was reversible on short time scales, and an equilibrium binding constant could be determined by SPR (KD = 1.31 μM), pointing out the only moderate affinity. However, inhibition was time-dependent, and 20 h of incubation with 98a abolished 50% of the apparent binding capacity of the slightly more potent saturated analogue NU6310 (98b, KD = 0.72 μM). Mass spectrometry and washout experiments confirmed the covalent modification. The binding to nonconserved Lys89 was suggested by studies with the CDK2 K89V mutant, which did not show adduct formation in MS experiments and recovered activity upon removal of the compound by dialysis. X-ray crystallography finally confirmed the binding mode (Figure 40B). The compound inhibited 13 out of 113 kinases in a panel at 1 μM but only two of those in a time-dependent manner. The rather slow inactivation kinetics might be rationalized by the surface-exposed location of Lys89, suggesting that the ε-amino group is almost completely protonated and therefore unreactive. However, it remains questionable if such slow inactivators would be useful in an in vivo setting as they only partially benefit from key advantages of covalent labeling (e.g., decoupling of pharmacodynamics from pharmacokinetics) while comparatively rapid off-target modification might be an issue, especially when quite reactive electrophiles are employed.

Figure 40

Figure 40. Vinyl sulfone targeting a lysine in the solvent-exposed front region of CDK2. (A) Covalent and noncovalent inhibitors. (B) X-ray crystal structure of vinyl sulfone 98a covalently bound to Lys89 flanking the solvent-exposed front region of CDK2 (PDB 5CYI). The purine NH is hydrogen-bonded to the backbone carbonyl atom of Glu81, while the N3-atom and the diaryl NH are anchored to the backbone of Leu83 by two additional hydrogen bonds. Another water-bridged hydrogen bond links the purine N7-atom to the backbone NH group of Asp145 in the DFG motif. Further direct and water-mediated hydrogen bonds are established by the sulfonyl group. The N-terminal lobe was omitted for clarity.

4.2. Reactivities of Sulfonyl Fluorides and Related Sulfur (VI) Fluorides toward Lysine, Tyrosine, and Cysteine

Sulfonyl fluorides are among the most prominent electrophiles for addressing the ε-amino group of lysines. They have also been used to target tyrosine, activated serine, or threonine residues and are reactive toward cysteine and histidine side chains as well.(265) The potential of sulfonyl fluorides, which have been known as insecticides for about 100 years,(266) in drug discovery was first recognized by Bernard Baker in the late 1960s.(267) However, until its recent revival by Sharpless and co-workers, sulfur (VI) fluoride chemistry (sulfonyl fluorides/chlorides are interpreted as sulfur (VI) halides in this context) has played a subordinate role while sulfonyl chlorides have frequently been used as reagents in organic synthesis.(266) The high reactivity of sulfonyl chlorides, as well as their instability toward hydrolysis and reduction, have hampered their use for biological applications. In contrast, sulfur (VI) fluorides are thermodynamically stable and less prone to hydrolysis and reduction. Unlike their chloride analogs, they are quite demanding electrophiles requiring sufficiently strong nucleophiles and proper solvation of the fluoride group for reaction. While sulfonyl chlorides may also be attacked on the chlorine atom, sulfonyl fluorides exclusively react at the sulfur atom. Therefore, sulfur (VI) fluoride exchange (SuFEx) has recently manifested itself as a useful click-type chemical reaction(266) with favorable properties for labeling biomolecules.(45) Sulfonimidoyl fluorides, the imino-analogues of sulfonyl fluorides, possess similar favorable properties and an additional handle to attach substituents, allowing for modulation of reactivity by the moiety attached to the nitrogen atom. Fluorosulfates and the analogous sulfamoyl fluorides are even less reactive (Figure 41). It has been further suggested that fluorosulfates only become activated if the departing fluoride ion is stabilized by the surrounding protein, e.g., by a hydrogen bond donor or suitable electric field effects.(268) Therefore, this functional group has been termed a “latent electrophile.”(269)

Figure 41

Figure 41. Sulfur (VI) fluorides for lysine and tyrosine targeting.

Early examples of the application of sulfonyl fluorides in chemical biology and drug discovery include protease labeling reagents such as benzenesulfonyl fluorides (99a,b, Figure 42)(270,271) and dansyl fluoride (100),(272) enzyme inhibitors exemplified by dihydrofolate reductase inhibitors (e.g., NSC 127755, 101) and nucleotide-derived probes (e.g., 5′-p-fluorosulfonylbenzoyl adenosine, 5′-FSBA, 102).(273) Sulfonyl fluorides have also been used to generate irreversible GPCR ligands,(274−276) and the tyrosine-targeted covalent adenosine A1 receptor antagonist DU172 (103) has recently enabled the crystal structure determination of the latter receptor.(277) Methanesulfonyl fluoride (MSF, 104) is known as an irreversible acetylcholinesterase (AChE) inhibitor for more than 50 years.(278) On the basis of the rationale that AChE in the CNS has a substantially lower resynthesis rate than in peripheral tissue, clinical phase I and II studies have been conducted assessing the potential of MSF in the treatment of Alzheimer’s disease.(279,280) MSF doses up to 0.18 mg/kg three times a week were well tolerated while effectuating the desired AChE inhibition in vivo and improving cognitive performance. Further applications of sulfonyl fluorides have been reviewed recently.(39,265,266,281)

Figure 42

Figure 42. Examples of sulfonyl fluorides applied in medicinal chemistry and chemical biology.

A detailed assessment of the reactivity of sulfonyl fluorides and analogous sulfur (VI) fluorides (Figure 43) was provided by Neil Grimster and colleagues from AstraZeneca.(282) They found that arylsulfonyl fluorides readily react with N-acetylcysteine (NAC) at pH 7.5. Reaction rates of most derivatives were slightly higher compared to analogous N-aryl acrylamides. However, the reactivity of sulfonyl fluorides could be tuned over a wider range with a variety of electron-withdrawing and electron-releasing groups (Figure 43A), the rate following a log–linear relationship to the Hammett parameter of the aryl substituent. The higher sensitivity to the electronic properties of the aromatic ring system compared to α,β-unsaturated amides can be rationalized by the direct attachment of the sulfonyl fluoride group. The substitution products, however, were unstable under the assay conditions, suggesting this functionality to be unsuitable for durable cysteine modification. This finding is not unexpected because the thiosulfonate S-esters formed by fluoride displacement are known to be unstable toward hydrolysis and react with thiols to form disulfide bonds (see the mechanism in Figure 43D).(283) In cells, an analogous reaction would predominantly produce glutathione disulfide (GSSG) due to the high intracellular GSH concentrations. It can also be assumed that thiosulfonate S-esters within protein binding sites would be cleaved by reductive sample preparation, emphasizing that cysteine labeling in living systems would be difficult to capture. The reactivity of sulfonyl fluorides toward N-acetyl tyrosine and N-α-acetyl lysine was considerably lower than toward NAC, but stable substitution products were obtained in both cases. While the initial reaction rate of phenylsulfonyl fluoride (105a) with N-acetyl tyrosine was 3.4 times decreased compared to NAC, a 10-fold slower reaction was observed for N-α-acetyl lysine at pH 7.5. Reaction rates increased substantially when the pH was increased, reaching a similar level for tyrosine and lysine at pH 10 (cysteine reacted too fast for rate determination at this pH). However, no reaction other than hydrolysis was observed for N-acetylated serine. Warhead hydrolysis (pH 7.5) was rapid for electron-deficient arylsulfonyl fluoride derivatives (t1/2 ≈ 5–15 min), while analogues bearing electron-donating substituents were stable up to several days. Nevertheless, hydrolysis was much slower than reaction with the tyrosine side chain. As shown for N-acetyl tyrosine, the vinyl sulfonyl fluoride 106 was 9 times less reactive than 105a with no concomitant 1,4-addition being observed. The benzylic derivative phenylmethylsulfonyl fluoride (PMSF, 107), a reagent commonly used in biochemistry to label active site serine residues or to prevent protein degradation in cell lysates,(284) was rapidly hydrolyzed, presumably due to its α-CH acidity favoring the reaction via a sulfene-like intermediate. Plasma stabilities reflected the trend from the hydrolysis assay with 4-dimethylamino derivative 105d showing a favorable half-life (t1/2 = 25.1 h). Clearance data was obtained for compounds 105bd (165, 24 and 170 × 10−6 μL/min, respectively) and several 5′-FSBA analogues (vide infra) in rat hepatocytes. In the latter series, metabolic clearance correlated rather with the lipophilicity of the compounds than with the Hammett parameters of the aryl substituents, suggesting that inherent warhead reactivity is not the key determinant of hepatic metabolism.

Figure 43

Figure 43. Compounds used for assessing the reactivity of sulfur (VI) fluorides toward different amino acids. (A) sulfonyl fluorides. (B) sulfonimidoyl fluorides. (C) aryl fluorosulfates. (D) Mechanism of the reaction between phenylsulfonyl fluoride and NAC.

A series of related sulfonimidoyl fluorides (represented by 108af, Figure 43B) with different substituents at the nitrogen atom was also probed for reactivity toward N-acetyl tyrosine. As expected, these compounds featured a lower reactivity compared to the analogous sulfonyl fluorides and electron withdrawing N-substituents were required for obtaining substitution products at the given conditions. Among the activated derivatives, N-acylated derivatives 108a,b reacted slightly faster than sulfonyl fluoride 105a, while the N-Boc analogue 108c reacted at a similar rate. N-Methylated analogue 108f showed no reaction under the same conditions. Interestingly, hydrolytic stability was increased compared to 105a even for the more activated derivatives, suggesting this structural class as superior warheads in terms of stability, versatility, and tunability. However, it should not remain unmentioned that electron-rich N-aryl derivatives (e.g., compound 108d), in contrast to p-CF3-phenyl-substituted analogue 108e, degraded rapidly, presumably via an oxidative decomposition pathway. NAC also reacted with the N-acylated derivatives to form unstable products while N-alkylated derivatives remained inert. Aryl fluorosulfates 109 and 110 (Figure 43C) were completely stable at pH 7.5, and neither hydrolysis nor substitution with N-α-acetyl lysine or tyrosine occurred within a 24 h time frame. Reaction with NAC was very slow, but extended incubation (4 days) resulted in the formation of the corresponding phenol, presumably via cystine formation in analogy to Figure 43D and subsequent loss of SO2. It is worth mentioning that the intrinsic reactivity of fluorosulfates is so low that fluorosulfate-l-tyrosine could recently be genetically encoded and incorporated into proteins as a latent electrophile for inter- and intraprotein cross-linking in mammalian cells.(285) No sulfamoyl fluorides were evaluated in this study, but it can be assumed that these compounds would even be less reactive than the corresponding aryl fluorosulfates.(266)

4.3. Lysine Targeting with Sulfur (VI) Fluorides

In the second part of the last-mentioned study, the reactivity of sulfonyl fluorides in a protein binding pocket was probed. To this end, analogues of the above-mentioned inhibitor 5′-FSBA (m-5′-FSBA; general structure 111, Figure 44) were prepared with different substituents ortho to the warhead. A crystal structure of prototype inhibitor 111a in complex with FGFR1 (PDB 5O49) and MS experiments confirmed the expected binding to the conserved Lys514 and suggested that an ortho-substituent could be accommodated by the binding site. The rate of covalent modification was evaluated by an LC-MS method showing that steric bulk in the ortho position did not significantly influence the reaction rates, which roughly correlated with the substituent’s Hammett parameters. As expected, two analogous aryl fluorosulfate derivatives showed a very low reactivity with only traces of covalent modification being detectable after extended reaction times.

Figure 44

Figure 44. Lysine-targeted m-5′-FSBA analogues for the evaluation of the relationship between warhead reactivity and FGFR1 inhibitory activity.

An interesting application of sulfonyl fluorides as CRGs was provided in 2017 by Jack Taunton and co-workers, who generated a lysine-targeted broad spectrum kinase inhibitor as an active-site probe for chemical proteomics applications.(286) A promiscuous pyrimidine 3-aminopyrazole scaffold was selected based on X-ray crystal structures of such ligands in the ATP pockets of various kinases. Structural data indicated that a suitable linker attached to the 2-position of the pyrimidine ring would position a phenylsulfonyl fluoride proximal to the conserved active site lysine, a residue that is involved in the positioning and activation of the ATP triphosphate moiety. Using SRC as a model kinase, it was shown by MS and X-ray crystallography that optimized and clickable probe XO44 (112, Figure 45A) specifically modified the expected lysine (Lys295, Figure 45B), while none of the other 16 lysines and 13 tyrosines were labeled by a 3-fold excess of the compound. Another X-ray crystal structure (PDB 5U8L) confirmed a similar binding mode for the EGFR receptor tyrosine kinase. In Jurkat T-cells, the inhibitor captured 133 protein kinases (of which 50 are not covered by the kinobead technology(287)) and even 219 kinases were inhibited >50% at 1 μM in a panel containing 375 kinases. The increased number of kinases captured in the panel could reflect the expression pattern of kinases in Jurkat cells. On the other hand, it might be an artifact from the nonphysiological conditions of in vitro screening panels or arise from noncovalent inhibition that cannot be captured in the cellular labeling experiment. Although some nonkinase off-targets were also modified, kinases accounted for the bulk of signal in MS experiments. Competition experiments with dasatinib subsequently validated the use of probe 112 for cellular selectivity profiling.

Figure 45

Figure 45. (A) Promiscuous kinase probe XO44. (B) X-ray crystal structure of XO44 covalently bound to the conserved Lys295 in the kinase SRC (PDB 5K9I). The 3-aminopyrazole is anchored to the hinge region by three hydrogen bonds involving the backbone of Glu339 and Met341. The sulfonyl group forms two additional hydrogen bonds with Phe278 and Gly279 in the glycine-rich loop, while the propargyl amide tag is oriented toward the bulk solvent without being involved in specific interactions.

As an application example of aryl fluorosulfates, these warheads were used by Jeffery Kelly and co-workers in fluorogenic probes to image transthyretin in living cells and Caenorhabditis elegans.(288) Cell permeable inhibitors 113a,b (Figure 46A) were designed to target the pKa-perturbed Lys15 side chain, which had previously been addressed with the analogous sulfonyl fluoride 114.(289) Although the probes were well-suited for the envisaged imaging purposes, covalent modification was very slow and only 6% and 29% labeling were observed after 24 h with 113a and 113b, respectively. These findings comply with the known low reactivity of the fluorosulfate group, however, the slow reaction could also be indicative of a suboptimal alignment between the reactants. Despite the slow reaction, MS-based analysis confirmed Lys15 as the site of modification while little general reactivity toward the proteome could be detected. Unexpectedly, the ε-amino group was present as a free sulfamate without the ligand attached (Figure 46B) indicating that the substitution product had been hydrolyzed. Although the authors stress the probable catalytic effect of the target protein on hydrolysis, these results demonstrate the limited stability of such covalent linkages that will have to be evaluated on a case-by-case basis. In proteins where the lysine residue is required for catalytic activity, the ε-sulfamate would still be detrimental to activity while one equivalent of the unreactive ligand with a free phenolic hydroxy group would be released. Further data on the general stability of protein-linked disubstituted sulfates or sulfamates under physiological conditions would be appreciated. Examples for the use of fluorosulfates in tyrosine and serine targeting can be found in the sections 5.1 and 6.1.

Figure 46

Figure 46. Fluorosulfates for lysine targeting. (A) Aryl fluorosulfates and sulfonyl fluorides designed for adressing Lys15 in human transthyretin. (B) X-ray crystal structure of 113b bound to human transthyretin (PDB 4YDM). Unexpectedly, the free Lys15 ε-sulfamate was observed instead of the covalently bound ligand. The ligand is located in a relatively shallow pocket on the protein surface forming only a single conserved hydrogen bond between the hydroxy group of the dichlorophenol moiety and the side chain of Ser117. An alternative orientation of the 3-hydroxyphenyl residue was omitted for clarity.

4.4. Lysine Acylation by Activated Esters

The potential of activated esters for targeting lysine side chains was recently emphasized by a team around Sebastien Campos at GSK.(290) With the aim of generating a potent and selective covalent PI3Kδ inhibitor, they modified the clinical candidate GSK2292767 (115, Figure 47), a highly optimized and isoform-selective reversible PI3Kδ inhibitor. The sulfonamide moiety in 115 had been shown to interact with the conserved Lys779 ε-amino group, suggesting that replacement by an activated ester (116af) would be a promising strategy to address this residue. A variety of phenolic esters with electron-withdrawing or releasing substituents in the para position was prepared and tested for their ability to covalently inactivate PI3Kδ. Isoform selectivity and activity in human whole blood were assessed in parallel. Activity in the isolated kinase assay correlated roughly with the electronic properties of the substituents. The 4-nitro derivative 116b proved to be most potent (pIC50 = 9.2) but featured a slightly decreased isoform selectivity and limited stability in DMSO. In contrast, slightly less reactive esters 116a,c,d maintained low nanomolar potency, high isoform selectivity (ca. 2–3 orders of magnitude), and excellent activity (≈ 10 nM) in human whole blood. Kinetic analysis revealed that the rate of covalent inactivation was similar for all compounds (kinact = 5.5–7.5 × 10–3 s–1) and did not correlate with the leaving group properties of the phenolate, while KI spanned a range from 40 nM (compound 116b) to 7.8 μM (compound 116f). Mass spectrometry and X-ray crystallography confirmed the exclusive labeling of Lys779 by 4-fluorophenyl ester 116d, and as expected, the nonactivated methyl ester 116g bound only reversibly to the enzyme. Compound 116d was stable against hydrolysis and reaction with N-α-Boc-lysine at pH 7.4. Interestingly, 116d showed a selectivity window in which PI3Kδ was covalently inhibited while PI3Kα and β engagement was reversible, indicating that covalent bond formation is critically dependent on reversible interactions. The compound showed high selectivity in a panel of 140 protein and 10 lipid kinases and the analogous azide-labeled probe 117 revealed a relatively clean chemoproteomic profile in Ramos cells. Washout experiments in CD4+ T-cells showed sustained suppression of IFNγ release for 48 h upon stimulation with αCD3, indicating prolonged cellular target engagement. However, no data on the metabolic stability of these compounds were reported. It is not unlikely that such activated esters are rapidly inactivated by esterases, which may erode activity in vivo or, following the rationale of Cravatt’s fumarates (section 2.1), improve kinetic selectivity. Finally, it should be noted that activated carbamates that have been frequently employed to target catalytic serines in drug discovery and crop protection(291) might be used in a similar manner as these activated esters.

Figure 47

Figure 47. Development of activated esters covalently targeting Lys779 in PI3Kδ.

4.5. Acylation of Surface-Exposed Lysines by N-Acyl-N-alkyl Sulfonamides

Another structure class that has recently been used to target lysines are N-acyl-N-alkyl sulfonamides. In early 2018, Itaru Hamachi and co-workers reported on the optimization of these moieties to achieve rapid ligand-directed labeling of surface-exposed lysine residues.(292) Activation of the acyl functionality was achieved by attaching electron-withdrawing groups to the sulfonamide nitrogen atom via a methylene spacer (Figure 48A). In an initial experiment, FKBP12 was used as a model protein. SLF, a known FKBP12 ligand (reported KD = 20 nM), was attached via a spacer to the sulfonamide sulfur atom while biotin was employed as the transferable N-acyl moiety and a cyanomethyl residue was utilized as the activating group (compound 118, KI= 210 nM). Incubation of this probe with FKBP12 predominantly biotinylated a single surface-exposed lysine, Lys44, located near the ligand binding site. Reasonable rates (kinact/KI = 2.9 × 104 M–1 s–1, kinact = 6.1 × 10–3 s–1) were achieved with this compound, while the replacement of the nitrile group by 4-nitrophenyl or 2,4-dinitrophenyl slowed down reaction kinetics substantially. Hydrolysis of compound 118 in aqueous buffer at pH 7.2 was slow, with a half-life of 43 h. The probe selectively labeled FKBP12 with biotin in HeLa cell lysates when the probe and the protein were present at an equimolar concentration (both 1 μM). However, increasing the probe concentration or competition with the high-affinity ligand rapamycin suppressed FKBP12 biotinylation and caused unspecific labeling. This underlines again the importance of potent and selective reversible binding for target specificity, especially when highly reactive electrophiles are employed. Remarkably, similar experiments were conducted with an analogous trimethoprim-derived probe (not shown) specifically labeling Lys32 in E. coli dihydrofolate reductase (kinact/KI = 9.3 × 103 M–1s–1, kinact = 1.3 × 10–2 s–1). In an inverse approach, an irreversible Hsp90 inhibitor (120, Figure 48B) was generated by using the known ligand PU-H71 (119) as the transferable N-acyl substituent. A spacer was required to bridge the distance between the target lysine and the ligand binding site. Compound 120 featured a KI of 62 nM and modified Lys58 (kinact/KI = 2.9 × 104 M–1 s–1) as determined by MS. The compound showed durable engagement of Hsp90 in SKBR3 cells, as demonstrated by washout and competition experiments. Encouragingly, an inverted probe featuring a transferable fluorescein marker labeled only four additional proteins, all of those to a lesser extent than Hsp90, indicating good cellular selectivity. Although labeling of surface-exposed lysines, which are typically poor nucleophiles, is a special feature of this compound class, this behavior could not be fully rationalized. Despite the reasonable reaction kinetics and the potential of further tuning by suitable N-alkyl substituents, the CRG is relatively bulky and might be usable only for special applications. For targeting surface-exposed lysine side chains, this bulkiness might not be a major issue, in sharp contrast to such lysines located in spatially restricted binding sites. Finally, despite the good cellular selectivity demonstrated for compound 120, the intrinsic reactivity of this warhead class toward other nucleophiles remains yet to be assessed.

Figure 48

Figure 48. N-Acyl-N-alkyl sulfonamides addressing surface-exposed lysine side chains. (A) Biotin-transferring probes. (B) Covalent ligand design and application example. The transferable residue is highlighted in red.

Further examples of electrophiles that have been used to irreversibly target lysine residues include natural product-derived spiro-epoxides addressing Lys100 in phosphoglycerate mutase 1 (PGAM1)(293,294) and quinazolin-4(3H)-one hydroxamate esters modifying nonactive site lysines in the bacterial serine endoprotease DegS.(295) Chemoselective methods for irreversible lysine modification, such as capture with diazonium terephthalates(296) or ortho-phthalaldehyde-amine condensations,(297) have recently emerged, but these can primarily be considered useful for bioconjugation applications rather than for specific covalent targeting in complex biological systems. The interested reader is referred to recent reviews.(41−43,46)

4.6. Condensation of Lysine with Aldehydes Forming Stabilized Schiff Bases

Schiff bases (imines), which are formed by the reversible condensation of an amine with an aldehyde or ketone, are quite common in biological systems. Prominent examples include pyridoxal phosphate (PLP)-dependent enzymes or the light sensitive GPCR rhodopsin, both binding their cofactors via Schiff base formation.(298) In medicinal chemistry, however, ketones and especially aldehydes are relatively unpopular. One of the main reasons for their avoidance is metabolic liability, as these chemical functionalities are prone to reduction by aldo-keto reductases and short-chain dehydrogenases/reductases or, in the case of aldehydes, oxidation to the corresponding acids by aldehyde dehydrogenases.(299) However, as highlighted in section 2.12, aldehydes can also be surprisingly stable. A recent study showed that metabolically labile aldehydes can be replaced by ketones whose metabolic stability, solubility and, to a lesser extent, the ketone–imine equilibrium can be tuned by the attachment of suitable residues.(199) This strategy furnished novel inhibitors of the cystine–glutamate antiporter, but covalent target engagement was not proven experimentally. Although Schiff base formation is highly reversible, mass-spectrometric analysis of the covalent complex is possible after reducing the Schiff base with borohydride to obtain a stable secondary amine. For example, this technique has been used to demonstrate the covalent binding of the umbelliferon-derived ligand 4μ8C with two distinct lysine residues (Lys509 and Lys907) in the inositol-requiring enzyme (IRE) 1.(200)
The stabilization of Schiff bases by coordination of the nitrogen lone pair to a boronic acid has been pursued by Pedro Gois and co-workers as a strategy for reversible protein modification.(300) In drug discovery, boronic acids are well-known as warheads for catalytic serine or threonine residues, as exemplified by the approved proteasome inhibitor bortezomib.(301) In this study, however, a boronic acid was introduced to the ortho-position of a benzaldehyde moiety in order to obtain an iminoboronate upon Schiff base formation. The latter is stabilized by an intramolecular dative bond between the nucleophilic nitrogen lone pair and the electrophilic boron center (Figure 49). In contrast to many of the above-mentioned methods, iminoboronate formation did not require pKa perturbation and proceeded readily between the model substrate 2-formylbenzeneboronic acid (121) and n-butyl amine at pH 6–9. The reaction was reversed upon the addition of GSH, fructose, and dopamine, suggesting reversibility under physiological conditions, which was later confirmed by Gao and co-workers.(302,303) Proteins such as cytochrome c, ribonuclease a, and myoglobin fully converted with excess 121 within 5 min.

Figure 49

Figure 49. 2-Formylbenzenboronic acid reversibly forming stabilized Schiff bases with amines.

Applications of this chemistry include the labeling of Gram-positive bacteria by targeting amine-presenting lipids(302) and more recently the inhibition of the induced myeloid leukemia cell differentiation protein (MCL-1), a protein–protein interaction target which is a key survival factor for various human cancers.(304) In the latter study, a group of researchers around Qibin Su and Neil Grimster attached a 2-carbonylphenylboronic acid warhead to a known indole-acid based MCL-1 inhibitor to reversibly target the surface-exposed Lys234 side chain. Formylphenylboronic acids 122a,b (Figure 50) as well as the analogous acetophenone 122c featured low nanomolar IC50 values in a TR-FRET assay, while derivatives lacking either the boronic acid or the ortho-carbonyl group were substantially less active. The improved potency translated into superior cellular activity, and the best compound (122b) showed an EC50 of 75 nM in the MCL-1-dependent cell line MOLP-8. Experiments with other MCL-1-dependent cell lines confirmed cellular activity, while MCL-1 independent cell lines remained unaffected. Mass spectrometry confirmed ca. 50% covalent labeling with 122a after 1 h, while acetophenone 122c reacted slightly slower. However, 50% conversion was not exceeded with an excess of 122a even after prolonged exposure, suggesting that an equilibrium stage had been reached. Reversibility was shown by SPR experiments and ligand dissociation fitted a two-state binding model (kd1 = 0.14 s–1 and 0.13 s–1; kd2 = 0.018 s–1 and 0.010 s–1 for 122a and 122b, respectively). It is worth mentioning that the warhead alone did not generate covalent complexes. Although no X-ray data was included, the modification of Lys234 was supported by binding data of covalent and noncovalent analogues to the K234A mutant. As demonstrated by this study, 2-carbonylphenylboronic acids can be used as cell-penetrable warheads that enable the reversible engagement of surface-exposed lysine side chains in the proximity of small molecule binding sites. It will be interesting to see whether addressing buried lysines, e.g., in kinases, will substantially decrease off-rates to enable complete labeling. Finally, the stability of this structure class under physiological conditions, especially with respect to the mentioned susceptibility of aldehydes and ketones toward redox biotransformation, will have to be assessed.

Figure 50

Figure 50. 2-Formylbenzenboronic acids and analogous acetophenones targeting MCL-1 via Schiff base formation.

5. Targeting the Tyrosine Side Chain

ARTICLE SECTIONS
Jump To

Neutral tyrosine has a relatively low intrinsic nucleophilicity when compared to cysteine or unprotonated lysine. Therefore, the selective modification of nonactivated tyrosine can be challenging. However, its phenolic hydroxy group possesses a slightly higher acidity compared to the protonated lysine side chain (pKa ≈ 9.7 vs 10.5),(305) and tyrosine residues in proteins can have a significantly lowered pKa, favoring the formation of the highly nucleophilic phenoxy anion.(306) Increased reactivity of tyrosine toward sulfonyl fluorides, for example, correlated with the proximity of a basic amino acid residue.(307) The phenoxy anion is a relatively hard nucleophile and favors the reaction with hard Lewis acids. Besides the nucleophilic properties of the phenolic hydroxy group, the electron-rich nature of the phenyl ring has been exploited for tyrosine-selective bioconjugation reactions, e.g., with in situ generated Mannich reagents,(308) diazonium salts,(309) or via ene-like reactions with triazoline diones.(310,311)

5.1. Tyrosine Targeting with Sulfur (VI) Fluorides

Because the inherent reactivity of tyrosine and lysine toward sulfur (VI) fluorides has already been discussed extensively in section 4.2, only application examples are provided in this section. Sulfonyl fluoride warheads have recently been used by Lyn Jones and colleagues from Pfizer to address the mRNA-decapping scavenger enzyme DcpS.(307) In a structure-based design approach, they modified known diaminoquinazoline-derived DcpS inhibitors to covalently address two proximal tyrosines, Tyr113 and 143, or the neighboring Lys142. The generated inhibitors featured low picomolar IC50 values in an ELISA-based DcpS activity assay (no binding kinetics were provided) and were approximately 2 orders of magnitude more potent than the reversible parent compound D153249 (123, Figure 51). X-ray and MS experiments showed that the ortho- and meta-substituted inhibitors 124a,b selectively modify Tyr113, while compound 124c with the CRG in the para-position reacts exclusively with Tyr143 (Figure 52). However, all of these inhibitors left Lys142 and His139 untouched, indicating that precise positioning of the warhead in conjunction with the appropriate intrinsic reactivity of the sulfonyl fluoride group for tyrosine confers specificity. It is worth mentioning that an analogous inhibitor with a silent click-tag revealed some off-targets that were not further disclosed.

Figure 51

Figure 51. Design of tyrosine-targeted sulfonyl fluorides as DcpS inhibitors.

Figure 52

Figure 52. Distinct binding modes of 124ac in the respective X-ray crystal structures in complex with DcpS. The ligand is completely embedded in the protein environment, and the 2,4-diaminquinazoline core adopts a similar orientation in all structures. Conserved interactions include two hydrogen bonds of the quinazoline 2-amino group to the carboxylate of Glu185 and the backbone carbonyl of Pro204 as well as a hydrogen bond between the quinazoline 4-amino group and the Asp205 side chain. The second proton of the quinazoline 4-amine forms an intramolecular H-bond to the ether linker. The (protonated) quinazoline N1-atom is also hydrogen-bonded to Glu185. (A) ortho-Substituted derivative 124a covalently bound to Tyr113 (PDB 4QDE). The phenyl ring points to the “bottom” of the binding pocket. Additional hydrogen bonds are formed to the side chains of Lys142 and Tyr273. (B) meta-Substituted derivative 124b covalently bound to Tyr113 (PDB 4QEB). Covalent attachment is enabled by an “upward” orientation of the phenyl ring, giving rise to an additional hydrogen bond between the sulfonyl group and the Tyr143 side chain. (C) para-Substituted derivative 124c covalently bound to Tyr143 (PDB 4QDV). The overall orientation resembles that of 124a, but Tyr143 is labeled instead of Tyr113. No hydrogen bonds with Lys142, Tyr273 and His139 are observed in (B) and (C), and the latter two residues were omitted for clarity. No covalent modification of the proximal nucleophiles Lys142 and His139 was observed in any of the experiments.

In a very recent study, Nathanael Gray and co-workers made use of a sulfonyl fluoride warhead to generate SRPKIN-1 (126, Figure 53), the first targeted kinase ligand covalently addressing a tyrosine residue.(312) While screening an internal compound library in the cellular KiNativ profiling assay,(313) the approved anaplastic lymphoma kinase (ALK) inhibitor alectinib (125)(314) was found to exhibit strong inhibitory activity toward the SR-protein kinase SRPK1 (IC50 = 11 nM on isolated SRPK1). A cocrystal structure gave insight into the binding mode of alectinib in complex with SRPK1, suggesting that replacement of the 4-morpholinopiperidine moiety by a meta-substituted phenyl ring bearing a sulfonyl fluoride warhead might enable covalent targeting of a unique tyrosine (Tyr227) adjacent to the solvent-exposed front region of the ATP binding site. Installation of this headgroup conserved most of the SRPK1 activity (IC50 = 36 nM) while rendering the compound a less potent ALK inhibitor (IC50 = 195 nM). No kinetic data was provided, thus the contribution of the covalent interaction to the observed potency remains unclear. Cellular KiNativ profiling demonstrated selectivity for SRPK1 and SRPK2, and washout experiments supported the anticipated covalent binding mode. Labeling of Tyr227 was subsequently confirmed by MS but no X-ray crystal structure was published. Notably, the compound suppressed neovascularization in a choroidal neovascularization model (CNV) after intravitreal injection in mice.

Figure 53

Figure 53. Design of sulfonyl fluoride SRPKIN-1, the first tyrosine-targeted covalent kinase inhibitor.

The less reactive aryl fluorosulfates have been suggested as privileged tyrosine-targeted electrophiles as they preferably react with phenolic hydroxy groups when compared to free alkanols, amines, thiols, guanidines, and heterocyclic NH groups.(315) Interesting work highlighting the special properties of aryl fluorosulfate warheads was published in 2016 by Kelly, Sharpless, Wilson, and colleagues. They showed that structurally simple aryl fluorosulfates can react specifically with tyrosine side chains in certain intracellular lipid binding proteins.(268) By using a clickable PEGylated phenyl fluorosulfate (127, Figure 54), it was demonstrated that this compound class selectively (and slowly) labels relatively few proteins as contrasted by an analogous sulfonyl fluoride. The cellular retinoic acid binding protein (CRABP) 2 was identified as the key target of 127 in HeLa cell proteomes. Rudimentary optimization furnished biphenyl-derived analogue 128 and fluorescent probe 129, enabling higher (but still moderate) CRABP2 labeling rates (kinact = 0.106 min–1 for 129). Structural comparison of CRABP2 with other lipid-binding proteins targeted by 127 suggested a spatially conserved Arg–Arg–Tyr carboxylic acid binding motif to be the key for covalent modification. Intriguingly, mutational depletion of either of the arginine residues impaired labeling. Studies at variable pH values indicated that the arginine side chain lowers the pKa of the tyrosine phenol moiety, and a simultaneous stabilization of the fluoride leaving group is likely. Cell-permeable probe 128 showed a very low background proteome labeling in HEK293T cells. An X-ray crystal structure in complex with CRABP2 (Figure 55) confirmed covalent bond formation with Tyr134. In contrast to the above-mentioned modification of transthyretin−Lys15 (section 4.3), the covalent linkage was stable. Finally, 128 was shown to inhibit CRABP-mediated delivery of retinoic acid to the nuclear retinoic acid receptor α in MCF-7 breast cancer cells. Notably, later studies indicated that aryl fluorosulfates have a low susceptibility to hepatic metabolism suggesting that such probes might also be applied in vivo.(316)

Figure 54

Figure 54. Aryl fluorosulfate probes targeting CRABP2 used in chemical proteomics studies.

Figure 55

Figure 55. X-ray crystal structure of the fluorosulfate-based ligand 128 covalently bound to Tyr134 in CRABP2 (PDB 5HZQ). The ligand is deeply buried in the binding site, and the sulfate group is engaged in a direct hydrogen bond to the Arg132 side chain and water-mediated hydrogen bonds to the Arg111 side chain. The PEG-linker is not resolved and a second, slightly deviating conformation of the ligand and the Tyr134 side chain was omitted for clarity.

Subsequently, the same groups reported on a strategy they denominated ″inverse drug discovery″, where the highly specific nature of latent fluorosulfate electrophiles was utilized to identify proteins with hotspots activating the latter for covalent lysine or tyrosine binding.(269) By capitalizing on three distinct alkyne-labeled probes of intermediate complexity (130132, Figure 56) in conjunction with unlabeled competitors, quantitative proteomics were employed to identify proteins that are efficiently labeled. In this approach, only a very low number of proteins was retrieved. This can be attributed to the very low reactivity of aryl fluorosulfates, however, the susceptibility of the reaction products to hydrolysis and elimination (vide infra), or the reaction with thiols to form unstable thiosulfate-S-esters (compare section 4.2) might also hamper product detection. Labeling at a specific site was validated for 11 targets by MS, mutagenesis, and X-ray crystallography using the recombinant proteins. As expected, covalent modification was only observed at tyrosine and lysine residues. Interestingly, probe 131 reacted with a largely different set of proteins compared to probes 130 and 132. Although no further optimization was performed, this study identified interesting targets that might be covalently addressed by sulfur (VI) chemistry. It should not remain unmentioned that the labeling sites could be reliably predicted by covalent docking which might be interesting for future drug discovery efforts.

Figure 56

Figure 56. Alkyne-tagged aryl fluorosulfate-based probes used in an “inverse drug discovery” approach.

5.2. Tyrosine Targeting by SNAr Reactions

LAS17 (133, Figure 57), an inhibitor with a 4,6-dichloro-1,3,5-triazine warhead, was recently shown by Lisa Crawford and Eranthie Weerapana to target Tyr108 in glutathione S-transferase π (GSTP1).(317) After having discovered by chemical proteomics that dichlorotriazines preferably label lysines while p-chloronitrobenzenes favor cysteines (see section 2.6),(130) a library of 20 N,N-disubstituted 4,6-dichloro-1,3,5-triazin-2-amines equipped with a click handle was synthesized and tested against HeLa cells at 1 μM. Protein enrichment revealed that one compound, LAS17, selectively modified a 25 kDa protein that was identified as GSTP1. This selectivity is quite surprising because 4,6-dichloro-1,3,5-triazin-2-amines chemically react with amino nucleophiles even at ambient temperature.(318) LAS17 inhibited GSTP1 in a concentration-dependent manner and activity was not comprised in the presence of the protein background from HeLa cell lysates. The second-order rate constant of inactivation (kinact/Ki) was determined as 3.12 × 104 M–1 s–1, and intact protein MS proved selective monolabeling. Further MS studies revealed Tyr108 as the only site of modification despite the presence of two highly reactive cysteine residues in GSTP1. It cannot be fully excluded that the reaction proceeds via an initial reaction with one of the more nucleophilic cysteines followed by transfer on Tyr108 to form the thermodynamically favored product. However, due to the distance between these residues (≥11 Å between the cysteines’ sulfur atoms and the phenolic oxygen in PDB 5X79) and their arrangement, such a low energy transfer pathway seems unlikely in this case. Mutation of Tyr108 to Phe prevented covalent modification and LAS17 possessed negligible activity on the mutant protein. It should be mentioned at this point that Tyr108 had previously been shown to be reactive toward sulfonyl fluoride-based probes.(319) Although this study highlights the ability of SNAr warheads to efficiently target tyrosine residues, it should be kept in mind that the employed electrophile features a high intrinsic reactivity. It is therefore remarkable that LAS17 only marginally labeled other proteins at a concentration of 1 μM emphasizing that intrinsic reactivity is not the only determinant for promiscuity and that the specificity and kinetics of reversible binding play a critical role for the selectivity of covalent inhibitors. In this context, further investigation of the specificity in cellular proteomes upon prolonged exposure to higher compound concentrations as well as a systematic evaluation of the minimal reactivity requirements for modifying Tyr108 with SNAr-type warheads would be very informative.

Figure 57

Figure 57. LAS17, a tyrosine-targeted dichlorotriazine-derived GSTP1 inhibitor.

6. Targeting Noncatalytic Serine and Threonine Residues

ARTICLE SECTIONS
Jump To

Serine and threonine are abundant as the key catalytic residues in the active sites of proteases and other hydrolase enzymes. At these locations, the side chain hydroxy groups are activated by neighboring residues, e.g., in catalytic triads, dramatically increasing their nucleophilicity.(320) A plethora of covalent inhibitors has been developed for these enzymes, and as mentioned already, many of the CRGs described in this Perspective had initially been employed to target the active sites of hydrolases.(7,321) A comprehensive discussion of covalent inhibitors of this enzyme class, however, is far beyond the scope of this article.
In contrast, noncatalytic serine and threonine residues are usually poorly acidic and therefore hardly reactive. Targeting these amino acids is still a significant challenge in bioconjugation chemistry. There is a very prominent example for the covalent modification of a noncatalytic serine by a drug, namely the acetylation of Ser530 in cyclooxygenases by aspirin.(322) Nevertheless, reports on compounds targeting noncatalytic serine and threonine residues in a proximity-driven manner remain very rare. In general, hard Lewis acids, such as sulfur (VI) fluorides or oxophilic phosphorus (V) compounds but also boron-based reagents, may be suited best for addressing such hydroxy groups.

6.1. Targeting Noncatalytic Serine by Fluorosulfates

In a follow-up of the study on diaminoquinazolines targeting tyrosine residues in DcpS (see section 5.1, Figure 51), Lyn Jones and co-workers sought to reduce off-target labeling by replacing the sulfonyl fluoride warheads by less reactive fluorosulfates.(316) Key compound FS-p1 (134a, Figure 58) featured increased stability toward hydrolysis and adequate membrane permeability. The turnover of this inhibitor in human liver microsomes was even lower as for the unsubstituted parent compound DAQ1 (134b), albeit at the expense of potency (IC50 = 3.2 nM) compared to the corresponding sulfonyl fluoride 124c (IC50 = <0.02 nM, cf. Figure 51). Unexpectedly, however, compound 134a modified neither of the expected tyrosine residues nor the adjacent lysine. Instead, reaction with the noncatalytic Ser272 was observed in peptide mapping experiments. LC-MS studies using the full protein and MS-based analysis after tryptic digestion did not reveal the labeled protein as the predominant species but the dehydroalanine (Dha) elimination product (see the mechanism in Figure 58B). In contrast, native ESI-MS(323) detected the labeled protein along with minor amounts of the dehydroalanine product, which slowly increased over time, suggesting that the latter species is mainly an artifact and not dominant in the protein’s binding site on a short time scale. The covalent engagement of Ser272 was especially surprising because this residue is not functionally relevant as the S272A mutant retains activity. The authors argued that the additional oxygen spacer would position the sulfur atom in an unfavorable position for being attacked by one of the more nucleophilic tyrosine or lysine side chains. The positive electrostatic nature of the binding pocket featuring an adjacent histidine triad may further depress the pKa of the serine and/or facilitate the departure of the fluoride leaving group. One implication of the limited stability of the sulfate diester products is that classical strategies for monitoring (off)-target modification, e.g., the click-chemistry based capturing approaches discussed before, would not be ideal for this warhead type. Alternative strategies will be required to examine the full scope of protein modification by such compounds in proteomes. Further investigation will also be required to show whether the formation of dehydroalanine-containing proteins could be a safety issue in vivo, as these are electrophilic themselves and may react with other physiological nucleophiles to generate hapten-carrier adducts. On the other hand, the underlying elimination reaction might be optimized and serve as a handle for specific protein derivatization.

Figure 58

Figure 58. (A) Aryl fluorosulfate-based inhibitor FS-p1 targeting Ser272 in DcpS. (B) Proposed mechanism for the formation of the dehydroalanine elimination product.

7. Targeting Glutamate and Aspartate Side Chains

ARTICLE SECTIONS
Jump To

Covalent modification of carboxylate residues as present in aspartate and glutamate side chains poses a special challenge due to the weakly nucleophilic nature of the (solvated) carboxylate group. Although carboxylates can react with many of the electrophiles discussed before (e.g., with epoxides or α-haloacetamides), these reactions are not specific to carboxylates and stronger nucleophiles react preferentially. Some approaches for the selective covalent trapping of carboxylates in proteins such as the photoclick-reaction with tetrazole-based reagents(324,325) or the reaction with α-diazo carboxamides(326) have been described as methods for bioconjugation. Cyclitol epoxides and aziridines as well as fluorinated glycosides have further found application as carboxylate-targeted electrophiles in activity-based probes and inhibitors of retaining glycosidases, an enzyme class featuring aspartate or glutamate residues as the catalytic nucleophile.(165) Sulfonate esters were shown to react preferentially with aspartate and glutamate in proteomes, but this behavior seems to be mediated by the respective protein environments as no such selectivity could be observed in solution.(157) On the other hand, it was shown that aziridines and stabilized diazo groups react with N-Boc-aspartate and benzoic acid in solution, but these CRGs failed to significantly label Asp12 in the K-Ras G12D mutant.(182) Boronic acids have further been suggested to interact with an aspartate in the front pocket of the EGFR kinase domain via reversible covalent bond formation,(327) but this interaction was not confirmed experimentally and is not specific for carboxylates. Hence, biocompatible warheads with a specific intrinsic reactivity for carboxylates are largely underdeveloped. However, selective and tunable CRGs for addressing this functionality would be particularly desirable because Asp and Glu constitute more than 10% of the protein sequence space(59) and are frequently found in or around binding pockets.

7.1. Targeting Carboxylates with Isoxazolium Salts

In a recent proof of concept study, Herbert Waldmann and colleagues employed isoxazolium salts derived from Woodward’s reagent K (WDK)(328) as site-specific covalent warheads for a glutamate residue in the lipoprotein binding chaperone phosphodiesterase (PDE) 6δ.(329) Because previous subnanomolar bis-sulfonamide-based inhibitors (exemplified by 135, Figure 59A) suffered from ligand displacement by allosteric release factor (ARL) 2/3 proteins,(330) covalent targeting was considered. Because no cysteine or lysine residues are available in the PDE6δ prenyl binding pocket, tosylate tags(331) were initially introduced for covalently addressing Glu88 but labeling efficiency was low. An alternative targeting strategy was inspired by Woodward’s isoxazolium-derived carboxylic acid activation reagents. N-Methyl isoxazolium salts (exemplified by general structure 136, Figure 59B) undergo ring opening in a base-promoted manner furnishing ketenimides (137), which readily react with carboxylates to form O-acylated 1,2-enolized β-ketoamides (138). The latter slowly rearrange to form the corresponding β-enol esters (139). It was hypothesized that initial ring opening could be initiated by proximal basic groups in the binding pocket, furnishing the reactive ketenimide species. Starting from 135, the benzyl moiety was replaced by a 4-unsubstituted N-methyl isoxazolium warhead attached via an ethylene spacer at the C5-position of the isoxazole ring (140a). The piperidine substituent was further replaced by a cyclohexyl ring in this series. As predicted, compound 140a and its homologue 140b covalently labeled PDE6δ at Glu88 as shown by mass spectrometry; covalent inactivation, however, was incomplete (80% after 30 min) and PDE6δ got reactivated within 24 h due to the limited stability of the reaction products. Shifting the attachment point to the C4-position of the isoxazole ring and introducing an additional methyl substituent at the C5-position (compounds 140c and 140d) increased labeling efficiency (>95% in less than 10 min) and only marginal cleavage was observed within 24 h. Finally, reintroduction of the piperidine nitrogen atom furnished 141, another promising compound from this series. Interestingly, removing the (piperidin-4-yl)methyl substituent from the sulfonamide nitrogen atom yielded a compound which did not effectuate any labeling pinpointing the critical role of reversible binding and accurate warhead positioning. Compound 140d possessed low reactivity toward the side chains of protected lysine and serine but cross-reacted with cysteine. However, no labeling of the PDE6δ E88A mutant was observed. As indicated by MS and fluorescence quenching studies, ARL proteins were unable to release the covalent ligands from the protein. Covalent attachment to Glu88 was further demonstrated by an X-ray crystal structure (Figure 60). It is worth noting that the ligand appears to be predominantly bound in the less stable form (compare structure 138). However, the electron density of the covalent tether is slightly less pronounced as for the rest of the ligand which might be attributed to partial conversion to the more stable β-enol ester product. Investigation of the selectivity of compound 141 using a cellular thermal shift assay (CETSA)(332,333) revealed only three off-targets (GDPGP1, PSMG3, PTGES2) along with PDE6δ.

Figure 59

Figure 59. Glutamate-targeted PDE6δ inhibitors. (A) Attachment of N-methyl isoxazolium warheads to reversible inhibitors exemplified by 135. (B) Mechanism of the reaction between the N-methyl isoxazolium group and carboxylates.

Figure 60

Figure 60. X-ray crystal structure of compound 140d covalently bound to Glu88 of PDE6δ (PDB 5NAL). The ligand is predominantly bound in the less stable O-acylated form and deeply buried in the binding site. Hydrogen bonds are formed by both oxygen atoms of the first sulfonyl group to the side chains of Arg61 and Gln78. An additional hydrogen bond is established between the second sulfonyl group and the side chain of Tyr149 (omitted for clarity).

The chemical stability of the conjugate against a 50-fold excess of hydroxylamine(334) suggests that the binding pocket shields the enol ester, thereby preventing the expected cleavage. It is likely that binding to surface-exposed carboxylate residues would be transient, and cleavage can also be expected after proteolytic degradation of the target protein, potentially reducing side effects by off-target binding and haptenization. However, whether these CRGs can be applied in living organisms remains to be proved. A general problem for in vivo applications might be found in the chemical stability of the warhead, which is good in acidic solution but only moderate at pH 7.4 and low at pH 9. Although this could offer a kinetic selectivity advantage, limited hydrolytic stability might be hardly compatible with oral dosing and the comparably slow distribution in vivo. It remains to be seen if reactivity tuning by modulating steric bulk and electronic properties of the substituents could resolve these issues. Although previous isoxazolium-derived activity-based protein profiling (ABPP) probes from the same group were able to penetrate cells,(335) it needs to be further investigated to what extent these charged molecules can cross biological barriers and how they are affected by metabolism in vivo. Finally, it will be interesting to see if other peptide coupling reagents might be tuned to become suitable carboxylate-targeted warheads for application in chemical probes or ultimately in drug discovery.

8. Targeting the Histidine Side Chain

ARTICLE SECTIONS
Jump To

Although the histidine imidazole group is frequently mentioned as a potential nucleophile for covalent targeting, only limited rational efforts have been described to address this amino acid. Histidine residues react with sulfonyl fluorides to form readily hydrolyzable sulfonylimidazoles as shown by the covalent modification of His130 in the active site of Salmonella typhimurium ribose-phosphate diphosphokinase by 5′-FSBA (102, Figure 42).(336) Moreover, histidine residues can undergo aza-Michael addition reactions with α,β-unsaturated ketones or aldehydes. For example, histidine residues were shown to react with 4-hydroxynonenal(337) or prostaglandin J2,(338) and more recently, a histidine of the vitamin D receptor has been covalently addressed with vitamin D-derived enones.(339)

8.1. Alkylation of Histidine by Spiro-epoxides

One of the prominent compounds addressing the histidine side chain is the natural product fumagillin (143, Figure 61) and the derived former clinical candidate beloranib (142), which bind to one of the active site histidines in methionine aminopeptidase (MetAP) 2 via opening of a spiro-epoxide (Figure 61B).(340) To improve the poor pharmacokinetics of this substance class, Aubry Miller and colleagues designed spiro-epoxytriazoles as drug-like fumagillin analogues (Figure 61C, general structures 144 and 145).(341) Several potent inhibitors of human MetAP2 were generated with key compound 145a (Figure 61D), featuring an IC50 value of 220 nM while its stereoisomers were inactive. As expected, enzyme inhibition was time-dependent and correlated well with cellular activities in human umbilical vein endothelial (HUVE) and HT1080 cells. Although the keto and benzoyl derivatives possessed poor stability in plasma and mouse liver microsomes, carbamate analogues were significantly more stable, even when compared with the former drug candidate beloranib. The labeling of His231 was confirmed by X-ray crystallography (Figure 62), while solvent-exposed nucleophiles (such as Cys290 and Lys427) remained untouched. These results indicate that histidine residues can be addressed specifically with relatively weak electrophiles, making them suitable targets for covalent inhibitor design. However, as for other moderate nucleophiles, the ligandability may largely depend on the precise positioning of the CRG and the target residues distinct nucleophilicity and thus on the surrounding protein environment.

Figure 61

Figure 61. Spiro-epoxides as histidine-targeted covalent inhibitors of hMetAP2. (A) Former drug candidate beloranib. (B) Reaction of the natural product fumagillin with His231 in hMetAP2. (C) Simplified fumagillin-derived structures. (D) hMetAP2 inhibitor 145a with improved PK properties.

Figure 62

Figure 62. X-ray crystal structure of the covalent complex between hMetAP2 and compound 145a (PDB 5CLS). His231 is covalently attached to the methylene group formed from the terminal carbon atom of the epoxide ring. The ensuing hydroxy group is linked to Asp251 and His382 by water-mediated hydrogen bonds. A direct hydrogen bond between the carbamate’s carbonyl group and the Asn329 backbone NH, and further water-mediated hydrogen bonds additionally anchor the ligand in the binding site.

8.2. Reversible Addition of Histidine to α-Cyanoenones

A covalent-reversible approach was recently pursued by Clarissa Jakob and co-workers from AbbVie to address a histidine in wild-type isocitrate dehydrogenase (IDH)1.(342) A high-throughput screen identified inhibitor 146a (Figure 63A) with an IC50 value of 410 nM. Optimization furnished the most potent carboxy-substituted analogue 146b (IC50 = 41 nM) and the slightly less active but cell permeable key compound 146c (IC50 = 110 nM). The latter inhibitor decreased reductive glutaminolysis in A498 cells in a dose-dependent manner, suggesting cellular target occupancy. Covalent modification of His315 in the NADPH binding pocket via aza-Michael addition to the α-cyanoenone moiety was confirmed by X-ray crystallography (Figure 63B), while no concomitant modification of exposed cysteines was observed. In accordance with the X-ray crystal structure showing a hydrogen bond between the backbone of Ser326 and the nitrile group, the removal of the α-cyano substituent in series 146 was detrimental to activity, while saturation of the double bond precluding covalent modification led to a moderate 16-fold loss in inhibitory potency. The latter finding suggests that the hydrogen bond to the nitrile group not only increases the inhibitors electrophilicity but is also crucial as a noncovalent key recognition element. Reversibility was demonstrated by wash out and jump dilution experiments. Consistent with transient covalent binding, no adducts were observed in mass spectrometric experiments.

Figure 63

Figure 63. α-Cyanoenones as histidine-targeted covalent-reversible IDH1 inhibitors. (A) Hit compound 146a and optimized derivatives. (B) X-ray crystal structure of 146c bound to IDH1 (PDB 6BL1). His315 is covalently attached to the β-position of the enone precursor. A key hydrogen bond is formed between the α-cyano moiety and the backbone NH of Se326. The enone keto group is hydrogen-bonded to the Lys374 side chain, while the diarylamino group forms a charge-assisted hydrogen bond to the carboxylate of Asp375.

9. Targeting Methionine Side Chains

ARTICLE SECTIONS
Jump To

Methionine is among the rarest amino acids in vertebrates and due to its high lipophilicity, typically buried within proteins. As mentioned before, the nucleophilicity of the methionine side chain is only moderate, however, its sulfur atom can be readily oxidized. Protocols for methionine labeling typically rely on highly reactive electrophiles at low pH.(343)

9.1. Redox-Activated Labeling of Methionine by Oxaziridines

An interesting new method for the selective labeling of methionine residues in proteins and cell lysates has recently been identified by the groups of Dean Toste and Christopher Chang.(343) They aimed to exploit the distinct redox activity of methionine for redox-activated chemical tagging (ReACT) under physiological conditions. A screening identified oxaziridines as suitable strain-driven sulfur imidation reagents.(344) Incorporating the oxaziridine nitrogen atom into a weakly electron-withdrawing urea moiety (exemplified by compound 147a, Figure 64A) avoided problems with concomitant sulfoxide formation observed for analogous carbamates (e.g., compound 147b). Sulfur imidation proceeds via a nucleophilic attack of the sulfur lone pair at the oxaziridine nitrogen atom followed by ring opening and release of benzylic aldehydes or ketones to furnish the S-imidation product (Figure 64B/C). The S-oxidation byproduct is formed in an analogous manner by attack at the oxygen atom. Labeling was selective for methionine over other amino acids (free cysteine and selenocysteine were oxidized to their cystine forms), and the products were relatively stable even in the presence of acid, base, and reducing agents. Alkynylated probe 147c at a 1 mM concentration incubated for 10 min with HeLa cell lysates labeled 235 methionine residues, and only a single lysine residue. Different concentrations of 147c showed a dose-dependent increase in the number of labeled residues allowing the identification of hyper-reactive methionines. The suitability of the approach for selective protein labeling and the construction of antibody–drug conjugates was also demonstrated. Despite their chemoselectivity, the presented oxaziridine-based probes are still highly reactive and convert methionine at rates similar to CuAAC reactions. It remains to be seen if steric shielding and/or tuning of electronic properties could furnish more specific warheads for chemical probe or drug design and if the bulkiness of such CRGs will limit their use with respect to the spatial constrains of many binding pockets. The underlying principle, however, is very promising and might stimulate research on similar approaches for specifically addressing methionine side chains.

Figure 64

Figure 64. Methionine-targeted oxaziridines. (A) Urea and carbamate-derived analogues. (B) Reaction with methionine via S-imidation or concomitant S-oxidation. Conditions: (1) D2O/CD3OD = 1:1, 2.5 min or (2) D2O/CD3OD = 95:5, 20 min. (C) Reaction mechanism of covalent methionine modification.

10. Targeting Other Amino Acids

ARTICLE SECTIONS
Jump To

It is not surprising that relatively few methods have been described to selectively address weak nucleophiles such as arginine, asparagine, glutamine, or tryptophan. For example, the bidentate nature of arginine’s guanidinium group has been exploited for reactions with glyoxal-derived reagents,(345) forming comparably stable cyclic products while the reaction with lysine or cysteine is highly reversible. However, the latter compound class is unlikely to find broad application in drug discovery due to metabolic liability and potential toxicity issues. A recent method has been described for metal-free tryptophan-selective bioconjugation in proteins.(346) Although being conceptually interesting, this method requires an organoradical reagent and sodium nitrite as an additive precluding applications in TCI design. Addressing phenylalanine or amino acids with nonactivated aliphatic side chains is even more challenging. Although catalysis-based approaches might be practicable for bioconjugation in vitro, such chemistries are difficult to realize in cells or even in vivo. Therefore, it is unlikely that these amino acids will be amenable to covalent targeting in a medicinal chemistry setting in the near future.

11. Summary and Perspective

ARTICLE SECTIONS
Jump To

Covalent targeting has become an extremely powerful tool in drug discovery and chemical biology and substantial effort has recently been put into developing or repurposing warheads for TCI design. An overview of the warhead classes discussed in this article and their key characteristics is provided in Table 2. The reader is referred to the respective sections and the literature cited therein for details.
Table 2. Overview of the Warhead Classes Discussed in This Article
a

Excluding catalytic nucleophiles. Data based on the studies discussed in the respective chapters.

b

L: Protein/peptide/amino acid (labeling or reactivity assay);. P: Protein (activity, binding affinity, kinact/KI). Y: Cell lysate (chemical proteomics study). C: Intact cell (functional assay or chemical proteomics study). I: In vivo (mammals or human). Data based on the reports discussed in the respective sections and additional searches in PubMed and the DrugBank.(146)

c

Application restricted to the catalytic cysteine of DUBs and related cysteine proteases so far.

d

Modified in solution but aziridines were unable to address Asp12 in the K-Ras G12D mutant.

e

Suggested by experiments with n-butylamine as a model nucleophile.

f

Forms unstable S-ester reaction products with cysteine.

g

Hemithioacetal formation with Cys is likely but rapidly reversible.

Despite the plethora of CRGs described in the current literature, selecting the right warhead for a specific application remains a nontrivial task. Especially in vivo, factors to be considered reach far beyond the structure of the protein and the nature of the target amino acid and include properties like target turnover, tissue distribution, and (sub)-cellular location, among others. Metabolic stability and chemical reactivity of the ligand also need to be well balanced, and the ideal window largely depends on the projected application. In this context, it is important to note that extrahepatic clearance is a major determinant of the pharmacokinetics of common acrylamide-derived TCIs.(347) Oxidative metabolism via reactive epoxide intermediates is another well-known biotransformation of α,β-unsaturated amides.(348) However, little is known about the metabolic fate of many CRGs discussed here. Reactivity toward glutathione, but also other thiol-containing reagents, has frequently been employed as a surrogate parameter to describe the nonspecific reactivity toward biological nucleophiles. In cells, however, much of the GSH-conjugation is mediated by glutathione S-transferases. Consequently, GSH addition under real physiological conditions might be much faster than estimated by the common (enzyme-free) GSH binding assays. An additional level of complexity is added by the presence of hyper-reactive cysteine residues in various proteins that could potentially engage even weakly reactive electrophiles. Similarly, certain features of protein binding sites might activate individual CRGs to become more electrophilic. In the light of this complexity, it should not remain unmentioned that some of the electrophiles discussed in this Perspective have only been evaluated in isolated proteins or peptides so far. Although these chemotypes may become useful in TCI design, the potential of such CRGs remains unclear until extensive profiling of reactivity, specificity, and stability in cells or in vivo has been performed.
Warhead selection typically starts with the estimation of reactivity required with respect to the desired target amino acid. Although computational reactivity prediction for certain electrophiles has significantly advanced within the last years,(12,158,349) precise and comparable rating over different warhead classed and nucleophiles remains a major challenge. When sufficiently exposed and reactive cysteines are to be addressed and slow depletion by GSH or other thiols is not an issue, α,β-unsaturated amides generally represent a safe bet. However, problems may arise when the target residue is poorly reactive, difficult to access, or incompatible with the spatial and geometric requirements of this electrophilic headgroup. Here, alternative acceptors, such as alkenylated or alkynylated heteroarenes, might come into play. SNAr warheads may offer advantages if spatially defined targeting needs to be combined with good metabolic stability and a highly tunable reactivity, especially if some additional steric bulk is not a problem. In the best case scenario, electron-deficient (hetero)arenes, which are already present in many biologically active compounds may simply be equipped with suitable leaving groups for covalently addressing proximal nucleophilic amino acids. Carbocyclic strain release warheads, which have a very low literature precedence, may be valuable alternatives especially if IP claims are an issue. Despite their promising chemical properties, however, the latter moieties have only been evaluated against isolated peptides so far. Systematic implementation of metabolically labile CRGs, such as fumaric acid esters or epoxides, is an option where short time exposure could offer a kinetic selectivity advantage. If reversible cysteine targeting is desired, α-cyanoacrylamides and analogous dually activated Michael acceptors are an up-to-date option with clinically approved representatives. Activated nitriles such as cyanamides or 2-cyanopyri(mi)dines represent suitable alternatives that have shown promising in vivo properties and aldehydes may also be employed in some cases.
Although the above-mentioned CRGs are typically used for cysteine targeting, many of them can also react with weaker nucleophiles such as lysine, tyrosine, or histidine. As discussed before, selective engagement of these less reactive residues is more challenging. Harder electrophiles, e.g., sulfonyl fluorides or activated esters, are more suitable to react with amino or hydroxy bases. Vinyl sulfones and the corresponding sulfonamides have further been shown to react readily with the lysine ε-amino group. Although the latter CRGs may be used for targeting lysines and tyrosines, most of these moieties have certain liabilities such as high intrinsic reactivity (vinyl sulfones and sulfonyl fluorides), limited stability toward hydrolysis (sulfonyl fluorides), and susceptibility to metabolic inactivation (activated esters). In the case of sulfur (VI) fluorides, the weakly reactive aryl fluorosulfates and the highly tunable sulfonimidoyl fluorides may address these issues and clearly merit further investigations. For reversible lysine targeting, 2-carbonylarylboronic acids forming stabilized Schiff bases may open up future avenues and further proof of concept, especially in vivo, would be highly desirable. However, despite these advances, the scope of lysine and tyrosine-targeted warheads is still limited and studies addressing the histidine side chain are even less abundant in the recent literature. Consequently, there is an urgent need for the development of novel CRGs which reliably and specifically address these residues without cross-reacting with cysteines.
Many weakly nucleophilic (e.g., glutamine, asparagine, arginine as well as nonactivated serine or threonine) or electrophilic (e.g., tryptophan) amino acids cannot be targeted reliably with the current warhead chemistry, necessitating further research to expand our scope of ligandable residues. Encouragingly, recent studies on glutamate/aspartate or methionine targeted CRGs hold the promise that at least some of the challenges associated with weaker nucleophiles could be addressed in the near future. Furthermore, the recognition of the role of cysteine oxidation in redox regulation has stimulated promising research on nucleophilic CRGs targeting electrophilic sulfenic acids and sulfenamides, which represent transient key oxidation products of cysteines’ thiols. Nevertheless, these concepts still have to prove their applicability in drug discovery and further refinement or alternative chemical approaches will be necessary when aiming for clinical applications.
Likewise, many issues with the more established warheads remain to be resolved. For example, none of the presented studies on sulfonyl fluorides, SNAr electrophiles, or activated esters investigated the toxic potential of the leaving group. Depending on the dose and the abundance/distribution of the target protein, it is conceivable that high local fluoride concentrations may result from fluoride-containing warheads in vivo. Similarly, phenols and other alcohols eliminated from activated esters could possess a distinct biological activity or toxicity themselves. Although these factors are not major issues for many chemical biology applications, they necessitate a detailed assessment prior to utilization in humans.
As it can be noticed throughout this article, IC50 values still dominate the literature on covalent ligands although the more laborious determination of binding kinetics is becoming more common. This reliance on IC50 data has implications on covalent ligand design because the observed differences in potency can be driven by reversible binding (KI) but also by the efficiency of covalent bond formation (kinact). In this context, it is interesting to note that the diffusion-based upper limit for the specificity constant (kcat/KM) of kinetically perfect enzymes is in the range of 108−109 M−1 s−1. Specific ligand association (described by kon), which is retarded, for example, by the requirement for pre-orientation or by induced fit, is usually slower and cannot exceed this cap.(34,350) Naturally, the same considerations apply to kinact/KI.
A well-designed TCI should be a potent and specific reversible binder to allow for selective target modification at low inhibitor concentrations. It should place the reactive moieties in favorable positions in terms of distance and angle. Similarly, the orientation toward activating residues in the binding cleft can play a role. The latter factors are crucial to enable a rapid bond formation between the reaction partners. The proper alignment of the reactive residues becomes especially important when low reactivity warheads are employed because the key properties of covalent binders (see the Introduction) would be lost with inactivation kinetics becoming too slow. In such a case, increased reversible potency could easily be mistaken as a result of covalent binding. Remarkably, recent data suggest that the efficacy of approved covalent EGFR inhibitors relies predominantly on reversible interactions.(219) Consequently, understanding binding kinetics is key to generate a properly balanced TCI which benefits from both the reversible and the covalent binding event.
To determine the specificity of novel CRGs and the derived TCIs in cells or even tissues, robust and convenient assays are required. Fortunately, considerable advances in chemical proteomics have made the assessment of off-targets in cellular proteomes becoming more and more common. Proteomic approaches provide very powerful tools to determine the fate of reactive ligands. Nevertheless, they also have their limitations. Methods employing tagged ligands to enrich covalently bound proteins, for example, may ignore the influence of the tag on ligand binding. On the other hand, approaches exploiting the differential labeling pattern of promiscuous reactive probes in the presence of a competitor ligand capture only a fraction of all potentially reactive amino acids present (e.g., the entire “cysteinome” or the “lysinome”) suggesting that relevant reaction sites could be missed. Even more of an issue are the intrinsic limitations of reactivity-based methods in identifying noncovalent interactions, which can significantly contribute to the biological profiles of reactive ligands. Many other issues are known and a further discussion may be found elsewhere.(351) Combination with emerging techniques, e.g., cellular thermal shift assays (CETSA)(332) or thermal proteome profiling,(333) and the ever increasing number of biochemical and cellular screening platforms, provides an opportunity to obtain a more complete picture. In this context, it is worth mentioning that little effort has been made so far to investigate the binding of reactive ligands to nonprotein off-targets, for example, the nucleobases in RNA and DNA. Such investigations would become even more important when employing more versatile warhead chemistries because each functional group features a distinct reactivity toward these nucleophiles. Altogether, understanding the fate of reactive ligands in complex living organisms still represents a fascinating challenge and a continuously developing area of research.
Ultimately, it seems unlikely that alternative warheads will replace cysteine-targeted α,β-unsaturated amides as the dominant chemical species in TCI design in the near future. Nevertheless, α,β-unsaturated amides are still far from being perfect warheads and do often not provide the general and predictable solutions they promise. Therefore, the CRGs presented here offer a valuable toolbox for the manifold applications where cysteine targeting with α,β-unsaturated amides cannot satisfy all needs or cases where simply no cysteine residues are available for covalent modification. Finally, we can expect a broadening of the scope of CRGs employed in drug discovery as challenging targets require the deliberate use of tailor-made warheads for success.

Author Information

ARTICLE SECTIONS
Jump To

  • Corresponding Author
  • Author
    • Stefan A. Laufer - Department of Pharmaceutical/Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, GermanyOrcidhttp://orcid.org/0000-0001-6952-1486
  • Notes
    The authors declare no competing financial interest.

Biographies

ARTICLE SECTIONS
Jump To

Matthias Gehringer

Matthias Gehringer studied chemistry at the Karlsruhe Institute of Technology, the Ecole Nationale Supérieure de Chimie de Montpellier, and the University of Heidelberg, and obtained his Ph.D. from Tübingen University working on reversible and irreversible kinase inhibitors. As a postdoctoral researcher at the Swiss Federal Institute of Technology (ETH) Zürich, he focused on the total synthesis of mycolactones and on targeted antibiotic–protein conjugates. He recently returned to Tübingen, where he is currently establishing an independent research group. His research interests include medicinal chemistry, chemical biology, natural-product synthesis, and innovative drug targeting approaches.

Stefan A. Laufer

Stefan A. Laufer studied Pharmacy and completed his Ph.D. from Regensburg University. After postdoctoral research in Frankfurt, he took a position in the pharmaceutical industry but maintained lectureships at Frankfurt and later at Mainz University, where he finished his habilitation in 1997. Since 1999, he has been full professor (chair) for pharmaceutical and medicinal chemistry at Tübingen University. He is cofounder/spokesman of ICEPHA (Interfaculty Center for Pharmacogenomics and Pharma Research), TüCADD (Tübingen Center for Academic Drug Discovery), and cofounder of the two startups CAIR Biosciences and Heparegenix. Three compounds from his lab made it first into man. He is currently (2016–2019) president of the German Pharmaceutical Society (DPhG). His research interests are protein kinase inhibitors and eicosanoid modulators.

Acknowledgments

ARTICLE SECTIONS
Jump To

We thank Dr. Michael Forster and Dr. Marcel Günther for fruitful discussions and Dr. Apirat Chaikuad for scientific advice. Kristine Schmidt, Dr. Michael Forster, Dr. Marcel Günther, and Bent Präfke are gratefully acknowledged for proof-reading. We thank Valentin Wydra and Nathanael Disch for assistance in the preparation of the manuscript and the TOC graphic. M.G. gratefully acknowledges financial support by the Institutional Strategy of the University of Tübingen (Deutsche Forschungsgemeinschaft, ZUK 63) and the Postdoctoral Fellowship Programme of the Baden-Württemberg Stiftung.

Abbreviations Used
ABPP

activity-based protein profiling

AChE

acetylcholinesterase

ADME

absorption, distribution, metabolism and excretion

ALDH2

aldehyde dehydrogenase 2

ALK

anaplastic lymphoma kinase

AR

androgen receptor

ARL

allosteric release factor

BTK

Bruton’s tyrosine kinase

CDK

cyclin-dependent kinase

CETSA

cellular thermal shift assay

CNV

choroidal neovascularization model

CRABP

cellular retinoic acid binding protein

CRG

covalent reactive group

CuAAC

copper(I)-catalyzed alkyne–azide cycloaddition

CYP

cytochrome P

DcpS

scavenger mRNA-decapping enzyme

DDAH

dimethylarginine dimethylaminohydrolase

DFT

density functional theory

Dha

dehydroalanine

DMF

dimethyl fumarate

DMPK

drug metabolism and pharmacokinetics

DMSO

dimethyl sulfoxide

DTT

dithiothreitol

DUBs

deubiquitinating isopeptidases

EC50

half-maximum effective concentration

EGFR

epidermal growth factor receptor

ErbB

protein family of four receptor tyrosine kinases (ErbB/Her 1–4)

ESI-MS

electrospray ionization-mass spectrometry

FDA

Food and Drug Administration

FSBA

5′-(4-fluorosulfonylbenzoyl)adenosine

FGFR

fibroblast growth factor receptor

FMK

fluoromethylketone

GAG

glycosaminoglycan

GK

gatekeeper

GPCR

G-protein coupled receptor

GPX

glutathione peroxidase

GSH

glutathione

GSTP1

glutathione S-transferase π or P1

H1975

human lung-cell line

hCES

human carboxylesterase

HCV

hepatitis C virus

HDAC8

histone deacetylase 8

HEK293

human embryonic kidney 293 cells

hERG

human ether-à-go-go-related gene

HSAB

hard and soft acids and bases

HTS

high throughput screening

HUVE

human umbilical vein endothelial

IC50

half-maximum inhibitory concentration

ICL

isocitrate lyase

IDH

isocitrate dehydrogenase

IRE

inositol-requiring enzyme

ITAM

immunoreceptor tyrosine-based activation motif

ITK

interleukin-2-inducible T-cell kinase

JAK

Janus kinase

K-Ras

p21 GTPase (oncogen first found in Kirsten rat sarcoma virus)

MAO

monoamine oxidase

MetAP

methionine aminopeptidase

MGMT

(O6-)methylguanine-DNA-methyltransferase

MMF

monomethyl fumarate

MSF

methanesulfonyl fluoride

MSBT

2-(methanesulfonyl)benzothiazole

MurA

UDP-N-acetylglucosamine enolpyruvyl transferase

NAC

N-acetylcysteine

NBD-dye

nitrobenzoxadiazole-dye

NCI

National Cancer Institute

NHS

N-hydroxysuccinimid

NMR

nuclear magnetic resonance

NS5B

nonstructural protein 5B

PDB

protein data bank

PDIA1

protein disulfide isomerase A1

PEG

polyethylene glycol

PEITC

phenethyl isothiocyanate

PGAM1

phosphoglycerate mutase 1

PLK

polo-like kinase

PLP

pyridoxal phosphate

PMBCs

peripheral blood mononuclear cells

PMSF

phenylmethane sulfonyl fluoride

PPARs

peroxisome proliferator-activated receptors

ReACT

redox-activated chemical tagging

ROS

reactive oxygen species

SAR

structure–activity relationship

SH

Scr homology

SNAr

nucleophilic aromatic substitution

SPR

surface plasmon resonance

SRC

steroid receptor coactivator

SRPK1

SR-protein kinase 1

SuFEx

sulfur (VI) fluoride exchange

SUMO

small ubiquitin-related modifier

TCEP

tris(2-carboxyethyl)phosphine

TCI

targeted covalent inhibitor

TM

transmembrane domain

TR

thyroid hormone receptor

TR-FRET

time-resolved Förster resonance energy transfer

TRPA1

transient receptor potential cation channel A1

Ub

ubiquitin

WDK

Woodward’s reagent K

References

ARTICLE SECTIONS
Jump To

This article references 351 other publications.

  1. 1
    Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nat. Biotechnol. 2003, 21 (3), 255261,  DOI: 10.1038/nbt0303-255
  2. 2
    Schopfer, F. J.; Cipollina, C.; Freeman, B. A. Formation and Signaling Actions of Electrophilic Lipids. Chem. Rev. 2011, 111 (10), 59976021,  DOI: 10.1021/cr200131e
  3. 3
    Allis, C. D.; Jenuwein, T. The Molecular Hallmarks of Epigenetic Control. Nat. Rev. Genet. 2016, 17 (8), 487500,  DOI: 10.1038/nrg.2016.59
  4. 4
    Uetrecht, J. Idiosyncratic Drug Reactions: Current Understanding. Annu. Rev. Pharmacol. Toxicol. 2007, 47 (1), 513539,  DOI: 10.1146/annurev.pharmtox.47.120505.105150
  5. 5
    Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307317,  DOI: 10.1038/nrd3410
  6. 6
    Bauer, R. A. Covalent Inhibitors in Drug Discovery: From Accidental Discoveries to Avoided Liabilities and Designed Therapies. Drug Discovery Today 2015, 20 (9), 10611073,  DOI: 10.1016/j.drudis.2015.05.005
  7. 7
    Powers, J. C.; Asgian, J. L.; Ekici, Ö. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. Chem. Rev. 2002, 102 (12), 46394750,  DOI: 10.1021/cr010182v
  8. 8
    Bachovchin, D. A.; Cravatt, B. F. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases. Nat. Rev. Drug Discovery 2012, 11 (1), 5268,  DOI: 10.1038/nrd3620
  9. 9
    Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. How to Improve R&D Productivity: The Pharmaceutical Industry’s Grand Challenge. Nat. Rev. Drug Discovery 2010, 9 (3), 203214,  DOI: 10.1038/nrd3078
  10. 10
    Bandyopadhyay, A.; Gao, J. Targeting Biomolecules with Reversible Covalent Chemistry. Curr. Opin. Chem. Biol. 2016, 34, 110116,  DOI: 10.1016/j.cbpa.2016.08.011
  11. 11
    Bradshaw, J. M.; McFarland, J. M.; Paavilainen, V. O.; Bisconte, A.; Tam, D.; Phan, V. T.; Romanov, S.; Finkle, D.; Shu, J.; Patel, V.; Ton, T.; Li, X.; Loughhead, D. G.; Nunn, P. A.; Karr, D. E.; Gerritsen, M. E.; Funk, J. O.; Owens, T. D.; Verner, E.; Brameld, K. A.; Hill, R. J.; Goldstein, D. M.; Taunton, J. Prolonged and Tunable Residence Time Using Reversible Covalent Kinase Inhibitors. Nat. Chem. Biol. 2015, 11 (7), 525531,  DOI: 10.1038/nchembio.1817
  12. 12
    Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57 (23), 1007210079,  DOI: 10.1021/jm501412a
  13. 13
    Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning, B. D.; González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F. Proteome-Wide Covalent Ligand Discovery in Native Biological Systems. Nature 2016, 534 (7608), 570574,  DOI: 10.1038/nature18002
  14. 14
    Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I. M.; Taunton, J. Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors. J. Am. Chem. Soc. 2013, 135 (14), 52985301,  DOI: 10.1021/ja401221b
  15. 15
    Jöst, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: A Systematic Study of Electrophilic Fragments. J. Med. Chem. 2014, 57 (18), 75907599,  DOI: 10.1021/jm5006918
  16. 16
    Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 2014, 57 (11), 49694974,  DOI: 10.1021/jm500345q
  17. 17
    Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M. K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions. Nature 2013, 503 (7477), 548551,  DOI: 10.1038/nature12796
  18. 18
    Zimmermann, G.; Rieder, U.; Bajic, D.; Vanetti, S.; Chaikuad, A.; Knapp, S.; Scheuermann, J.; Mattarella, M.; Neri, D. A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library. Chem. - Eur. J. 2017, 23 (34), 81528155,  DOI: 10.1002/chem.201701644
  19. 19
    Zambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N. Screening for Covalent Inhibitors Using DNA-Display of Small Molecule Libraries Functionalized with Cysteine Reactive Moieties. MedChemComm 2016, 7 (7), 13401351,  DOI: 10.1039/C6MD00242K
  20. 20
    Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discov. 2017, 22 (1), 320,  DOI: 10.1177/1087057116671509
  21. 21
    Miyahisa, I.; Sameshima, T.; Hixon, M. S. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (Kinact/Ki) by Means of an Endpoint Competition Assay. Angew. Chem., Int. Ed. 2015, 54 (47), 1409914102,  DOI: 10.1002/anie.201505800
  22. 22
    Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry. Annu. Rev. Biochem. 2008, 77 (1), 383414,  DOI: 10.1146/annurev.biochem.75.101304.124125
  23. 23
    Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase Inhibitors. Nat. Chem. Biol. 2014, 10 (9), 760767,  DOI: 10.1038/nchembio.1582
  24. 24
    Zaro, B. W.; Whitby, L. R.; Lum, K. M.; Cravatt, B. F. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors. J. Am. Chem. Soc. 2016, 138 (49), 1584115844,  DOI: 10.1021/jacs.6b10589
  25. 25
    Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen, M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Frödin, M.; Taunton, J. Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles. Nat. Chem. Biol. 2012, 8 (5), 471476,  DOI: 10.1038/nchembio.925
  26. 26
    Noe, M. C.; Gilbert, A. M. Targeted Covalent Enzyme Inhibitors. In Annual Reports in Medicinal Chemistry; Desai, M. C., Ed.; Academic Press, 2012; Vol. 47, pp. 413439.  DOI: 10.1016/B978-0-12-396492-2.00027-8 .
  27. 27
    Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20 (2), 146159,  DOI: 10.1016/j.chembiol.2012.12.006
  28. 28
    Miller, R. M.; Taunton, J. Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors. In Methods in Enzymology; Shokat, K. M., Ed.; Academic Press, 2014; Vol. 548, pp 93116.  DOI: 10.1016/B978-0-12-397918-6.00004-5 .
  29. 29
    Gilbert, A. M. Recent Advances in Irreversible Kinase Inhibitors. Pharm. Pat. Anal. 2014, 3 (4), 375386,  DOI: 10.4155/ppa.14.24
  30. 30
    Adeniyi, A. A.; Muthusamy, R.; Soliman, M. E. New Drug Design with Covalent Modifiers. Expert Opin. Drug Discovery 2016, 11 (1), 7990,  DOI: 10.1517/17460441.2016.1115478
  31. 31
    Baillie, T. A. Targeted Covalent Inhibitors for Drug Design. Angew. Chem., Int. Ed. 2016, 55 (43), 1340813421,  DOI: 10.1002/anie.201601091
  32. 32
    Hallenbeck, K. K.; Turner, D. M.; Renslo, A. R.; Arkin, M. R. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery. Curr. Top. Med. Chem. 2016, 17, 415,  DOI: 10.2174/1568026616666160719163839
  33. 33
    Lagoutte, R.; Patouret, R.; Winssinger, N. Covalent Inhibitors: An Opportunity for Rational Target Selectivity. Curr. Opin. Chem. Biol. 2017, 39, 5463,  DOI: 10.1016/j.cbpa.2017.05.008
  34. 34
    De Cesco, S.; Kurian, J.; Dufresne, C.; Mittermaier, A. K.; Moitessier, N. Covalent Inhibitors Design and Discovery. Eur. J. Med. Chem. 2017, 138, 96114,  DOI: 10.1016/j.ejmech.2017.06.019
  35. 35
    Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The Cysteinome of Protein Kinases as a Target in Drug Development. Angew. Chem., Int. Ed. 2018, 57 (16), 43724385,  DOI: 10.1002/anie.201707875
  36. 36
    Zhao, Z.; Bourne, P. E. Progress with Covalent Small-Molecule Kinase Inhibitors. Drug Discovery Today 2018, 23 (3), 727735,  DOI: 10.1016/j.drudis.2018.01.035
  37. 37
    Lonsdale, R.; Ward, R. A. Structure-Based Design of Targeted Covalent Inhibitors. Chem. Soc. Rev. 2018, 47 (11), 38163830,  DOI: 10.1039/C7CS00220C
  38. 38
    Ferguson, F. M.; Gray, N. S. Kinase Inhibitors: The Road Ahead. Nat. Rev. Drug Discovery 2018, 17 (5), 353377,  DOI: 10.1038/nrd.2018.21
  39. 39
    Pettinger, J.; Jones, K.; Cheeseman, M. D. Lysine-Targeting Covalent Inhibitors. Angew. Chem., Int. Ed. 2017, 56 (48), 1520015209,  DOI: 10.1002/anie.201707630
  40. 40
    Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60 (3), 839885,  DOI: 10.1021/acs.jmedchem.6b00788
  41. 41
    Baslé, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17 (3), 213227,  DOI: 10.1016/j.chembiol.2010.02.008
  42. 42
    Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 21742195,  DOI: 10.1021/cr500399p
  43. 43
    Shannon, D. A.; Weerapana, E. Covalent Protein Modification: The Current Landscape of Residue-Specific Electrophiles. Curr. Opin. Chem. Biol. 2015, 24, 1826,  DOI: 10.1016/j.cbpa.2014.10.021
  44. 44
    Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529553,  DOI: 10.1002/cbic.201500667
  45. 45
    Dondoni, A.; Marra, A. SuFEx: A Metal-Free Click Ligation for Multivalent Biomolecules. Org. Biomol. Chem. 2017, 15 (7), 15491553,  DOI: 10.1039/C6OB02458K
  46. 46
    deGruyter, J. N.; Malins, L. R.; Baran, P. S. Residue-Specific Peptide Modification: A Chemist’s Guide. Biochemistry 2017, 56 (30), 38633873,  DOI: 10.1021/acs.biochem.7b00536
  47. 47
    Hoch, D. G.; Abegg, D.; Adibekian, A. Cysteine-Reactive Probes and Their Use in Chemical Proteomics. Chem. Commun. 2018, 54 (36), 45014512,  DOI: 10.1039/C8CC01485J
  48. 48
    Cromm, P. M.; Crews, C. M. The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug Target. ACS Cent. Sci. 2017, 3 (8), 830838,  DOI: 10.1021/acscentsci.7b00252
  49. 49
    Casimiro-Garcia, A.; Trujillo, J. I.; Vajdos, F.; Juba, B.; Banker, M. E.; Aulabaugh, A.; Balbo, P.; Bauman, J.; Chrencik, J.; Coe, J. W.; Czerwinski, R.; Dowty, M.; Knafels, J. D.; Kwon, S.; Leung, L.; Liang, S.; Robinson, R. P.; Telliez, J.-B.; Unwalla, R.; Yang, X.; Thorarensen, A. Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors. J. Med. Chem. 2018, 61, 1066510699,  DOI: 10.1021/acs.jmedchem.8b01308
  50. 50
    Li, T.; Maltais, R.; Poirier, D.; Lin, S.-X. Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide. J. Phys. Chem. Lett. 2018, 9 (18), 52755280,  DOI: 10.1021/acs.jpclett.8b02225
  51. 51
    Kharenko, O. A.; Patel, R. G.; Brown, S. D.; Calosing, C.; White, A.; Lakshminarasimhan, D.; Suto, R. K.; Duffy, B. C.; Kitchen, D. B.; McLure, K. G.; Hansen, H. C.; van der Horst, E. H.; Young, P. R. Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine. J. Med. Chem. 2018, 61 (18), 82028211,  DOI: 10.1021/acs.jmedchem.8b00666
  52. 52
    Pearson, R. G.; Sobel, H. R.; Songstad, J. Nucleophilic Reactivity Constants toward Methyl Iodide and Trans-Dichlorodi(Pyridine)Platinum(II). J. Am. Chem. Soc. 1968, 90 (2), 319326,  DOI: 10.1021/ja01004a021
  53. 53
    Sardi, F.; Manta, B.; Portillo-Ledesma, S.; Knoops, B.; Comini, M. A.; Ferrer-Sueta, G. Determination of Acidity and Nucleophilicity in Thiols by Reaction with Monobromobimane and Fluorescence Detection. Anal. Biochem. 2013, 435 (1), 7482,  DOI: 10.1016/j.ab.2012.12.017
  54. 54
    Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc. 1963, 85 (22), 35333539,  DOI: 10.1021/ja00905a001
  55. 55
    Awoonor-Williams, E.; Rowley, C. N. Evaluation of Methods for the Calculation of the PKa of Cysteine Residues in Proteins. J. Chem. Theory Comput. 2016, 12 (9), 46624673,  DOI: 10.1021/acs.jctc.6b00631
  56. 56
    Grimsley, G. R.; Scholtz, J. M.; Pace, C. N. A Summary of the Measured PK Values of the Ionizable Groups in Folded Proteins. Protein Sci. 2008, 18, 247251,  DOI: 10.1002/pro.19
  57. 57
    Alcock, L. J.; Perkins, M. V.; Chalker, J. M. Chemical Methods for Mapping Cysteine Oxidation. Chem. Soc. Rev. 2018, 47 (1), 231268,  DOI: 10.1039/C7CS00607A
  58. 58
    Pace, N. J.; Weerapana, E. Diverse Functional Roles of Reactive Cysteines. ACS Chem. Biol. 2013, 8 (2), 283296,  DOI: 10.1021/cb3005269
  59. 59
    Jones, A.; Zhang, X.; Lei, X. Covalent Probe Finds Carboxylic Acid. Cell Chem. Biol. 2017, 24 (5), 537539,  DOI: 10.1016/j.chembiol.2017.05.003
  60. 60
    Shirley, M. Dacomitinib: First Global Approval. Drugs 2018, 78, 1947,  DOI: 10.1007/s40265-018-1028-x
  61. 61
    Markham, A.; Dhillon, S. Acalabrutinib: First Global Approval. Drugs 2018, 78 (1), 139145,  DOI: 10.1007/s40265-017-0852-8
  62. 62
    Forster, M.; Gehringer, M.; Laufer, S. A. Recent Advances in JAK3 Inhibition: Isoform Selectivity by Covalent Cysteine Targeting. Bioorg. Med. Chem. Lett. 2017, 27 (18), 42294237,  DOI: 10.1016/j.bmcl.2017.07.079
  63. 63
    Garzón, B.; Oeste, C. L.; Díez-Dacal, B.; Pérez-Sala, D. Proteomic Studies on Protein Modification by Cyclopentenone Prostaglandins: Expanding Our View on Electrophile Actions. J. Proteomics 2011, 74 (11), 22432263,  DOI: 10.1016/j.jprot.2011.03.028
  64. 64
    Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60 (7), 28792889,  DOI: 10.1021/acs.jmedchem.6b01815
  65. 65
    Günther, M.; Juchum, M.; Kelter, G.; Fiebig, H.; Laufer, S. Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant. Angew. Chem., Int. Ed. 2016, 55 (36), 1089010894,  DOI: 10.1002/anie.201603736
  66. 66
    Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.; Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem. Biol. 2017, 24, 13881400,  DOI: 10.1016/j.chembiol.2017.08.017
  67. 67
    Blewett, M. M.; Xie, J.; Zaro, B. W.; Backus, K. M.; Altman, A.; Teijaro, J. R.; Cravatt, B. F. Chemical Proteomic Map of Dimethyl Fumarate–Sensitive Cysteines in Primary Human T Cells. Sci. Signaling 2016, 9 (445), rs10,  DOI: 10.1126/scisignal.aaf7694
  68. 68
    Deeks, E. D. Ibrutinib: A Review in Chronic Lymphocytic Leukaemia. Drugs 2017, 77 (2), 225236,  DOI: 10.1007/s40265-017-0695-3
  69. 69
    Bender, A. T.; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.; Grenningloh, R.; Head, J.; Morandi, F.; Haselmayer, P.; Liu-Bujalski, L. Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but Not B-Cell Receptor Signaling. Mol. Pharmacol. 2017, 91 (3), 208219,  DOI: 10.1124/mol.116.107037
  70. 70
    Pan, Z.; Scheerens, H.; Li, S.-J.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase. ChemMedChem 2007, 2 (1), 5861,  DOI: 10.1002/cmdc.200600221
  71. 71
    Mann, M. Innovations: Functional and Quantitative Proteomics Using SILAC. Nat. Rev. Mol. Cell Biol. 2006, 7 (12), 952958,  DOI: 10.1038/nrm2067
  72. 72
    Crow, J. A.; Bittles, V.; Borazjani, A.; Potter, P. M.; Ross, M. K. Covalent Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by the Carbamates JZL184 and URB597. Biochem. Pharmacol. 2012, 84 (9), 12151222,  DOI: 10.1016/j.bcp.2012.08.017
  73. 73
    Buynak, J. D.; Mathew, J.; Rao, M. N.; Haley, E.; George, C.; Siriwardane, U. The Preparation of the First α-Vinylidene-β-Lactams. J. Chem. Soc., Chem. Commun. 1987, 0 (10), 735737,  DOI: 10.1039/C39870000735
  74. 74
    Roedig, A.; Ritschel, W. Reaktionen von 3,4,4-Trichlor-3-butenamiden mit Nucleophilen, II. Thiol- und Aminaddukte von 3,3-Dichlorallencarboxamiden. Chem. Ber. 1983, 116 (4), 15951602,  DOI: 10.1002/cber.19831160434
  75. 75
    Abbas, A.; Xing, B.; Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem., Int. Ed. 2014, 53 (29), 74917494,  DOI: 10.1002/anie.201403121
  76. 76
    Pedzisa, L.; Li, X.; Rader, C.; Roush, W. R. Assessment of Reagents for Selenocysteine Conjugation and the Stability of Selenocysteine Adducts. Org. Biomol. Chem. 2016, 14 (22), 51415147,  DOI: 10.1039/C6OB00775A
  77. 77
    Chen, D.; Guo, D.; Yan, Z.; Zhao, Y. Allenamide as a Bioisostere of Acrylamide in the Design and Synthesis of Targeted Covalent Inhibitors. MedChemComm 2018, 9 (2), 244253,  DOI: 10.1039/C7MD00571G
  78. 78
    Awoonor-Williams, E.; Rowley, C. N. How Reactive Are Druggable Cysteines in Protein Kinases?. J. Chem. Inf. Model. 2018, 58 (9), 19351946,  DOI: 10.1021/acs.jcim.8b00454
  79. 79
    Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J.-S.; Strub, J.-M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles. Bioconjugate Chem. 2014, 25 (2), 202206,  DOI: 10.1021/bc400469d
  80. 80
    Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.; Liu, Y.; Wong, M.-K.; Che, C.-M. Electron-Deficient Alkynes as Cleavable Reagents for the Modification of Cysteine-Containing Peptides in Aqueous Medium. Chem. - Eur. J. 2009, 15 (15), 38393850,  DOI: 10.1002/chem.200800669
  81. 81
    Friedman, M.; Wall, J. S. Additive Linear Free-Energy Relationships in Reaction Kinetics of Amino Groups with α,β-Unsaturated Compounds. J. Org. Chem. 1966, 31 (9), 28882894,  DOI: 10.1021/jo01347a036
  82. 82
    Cavins, J. F.; Friedman, M. An Internal Standard for Amino Acid Analyses: S-β-(4-Pyridylethyl)-l-Cysteine. Anal. Biochem. 1970, 35 (2), 489493,  DOI: 10.1016/0003-2697(70)90211-3
  83. 83
    Gill, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr, M. G.; Woodhead, A. J.; Walker, M. T.; Congreve, M. S.; Devine, L. A.; Tisi, D.; O’Reilly, M.; Seavers, L. C. A.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A. E.; Jhoti, H. Identification of Novel P38α MAP Kinase Inhibitors Using Fragment-Based Lead Generation. J. Med. Chem. 2005, 48 (2), 414426,  DOI: 10.1021/jm049575n
  84. 84
    Raux, E.; Klenc, J.; Blake, A.; Sączewski, J.; Strekowski, L. Conjugate Addition of Nucleophiles to the Vinyl Function of 2-Chloro-4-Vinylpyrimidine Derivatives. Molecules 2010, 15 (3), 19731984,  DOI: 10.3390/molecules15031973
  85. 85
    Burns, A. R.; Kerr, J. H.; Kerr, W. J.; Passmore, J.; Paterson, L. C.; Watson, A. J. B. Tuned Methods for Conjugate Addition to a Vinyl Oxadiazole; Synthesis of Pharmaceutically Important Motifs. Org. Biomol. Chem. 2010, 8 (12), 27772783,  DOI: 10.1039/c001772h
  86. 86
    Kuchař, M.; Hocek, M.; Pohl, R.; Votruba, I.; Shih, I.; Mabery, E.; Mackman, R. Synthesis, Cytostatic, and Antiviral Activity of Novel 6-[2-(Dialkylamino)Ethyl]-, 6-(2-Alkoxyethyl)-, 6-[2-(Alkylsulfanyl)Ethyl]-, and 6-[2-(Dialkylamino)Vinyl]Purine Nucleosides. Bioorg. Med. Chem. 2008, 16 (3), 14001424,  DOI: 10.1016/j.bmc.2007.10.063
  87. 87
    Il’yasov, E. A.; Galust’yan, G. G. Homolytic Addition of 1-Alkanethiols to 5-Ethynyl-2-Methylpyridine. Chem. Heterocycl. Compd. 1999, 35 (10), 11871189,  DOI: 10.1007/BF02323377
  88. 88
    Wipf, P.; Graham, T. H. Synthesis and Hetero-Michael Addition Reactions of 2-Alkynyl Oxazoles and Oxazolines. Org. Biomol. Chem. 2005, 3 (1), 3135,  DOI: 10.1039/b413604g
  89. 89
    Li, Q.-F.; Yang, Y.; Maleckis, A.; Otting, G.; Su, X.-C. Thiol–Ene Reaction: A Versatile Tool in Site-Specific Labelling of Proteins with Chemically Inert Tags for Paramagnetic NMR. Chem. Commun. 2012, 48 (21), 27042706,  DOI: 10.1039/c2cc17900h
  90. 90
    Yang, Y.; Li, Q.-F.; Cao, C.; Huang, F.; Su, X.-C. Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study. Chem. - Eur. J. 2013, 19 (3), 10971103,  DOI: 10.1002/chem.201202495
  91. 91
    Ma, F.-H.; Chen, J.-L.; Li, Q.-F.; Zuo, H.-H.; Huang, F.; Su, X.-C. Kinetic Assay of the Michael Addition-Like Thiol–Ene Reaction and Insight into Protein Bioconjugation. Chem. - Asian J. 2014, 9 (7), 18081816,  DOI: 10.1002/asia.201402095
  92. 92
    Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.; Dickerson, S. H.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell, A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S. M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J. D.; White, W. L.; Uehling, D. E. 6-Ethynylthieno[3,2-d]- and 6-Ethynylthieno[2,3-d]Pyrimidin-4-Anilines as Tunable Covalent Modifiers of ErbB Kinases. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (8), 27732778,  DOI: 10.1073/pnas.0708281105
  93. 93
    Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A. Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth Factor Receptor:  4-(Phenylamino)Quinazoline- and 4-(Phenylamino)Pyrido[3,2-d]Pyrimidine-6-Acrylamides Bearing Additional Solubilizing Functions. J. Med. Chem. 2000, 43 (7), 13801397,  DOI: 10.1021/jm990482t
  94. 94
    Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.; Wissner, A. 6-Substituted-4-(3-Bromophenylamino)Quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor Activity. J. Med. Chem. 2001, 44 (17), 27192734,  DOI: 10.1021/jm0005555
  95. 95
    Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, H.-R. Synthesis and Structure–Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-Carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2). J. Med. Chem. 2003, 46 (1), 4963,  DOI: 10.1021/jm020241c
  96. 96
    Hubbard, R. D.; Dickerson, S. H.; Emerson, H. K.; Griffin, R. J.; Reno, M. J.; Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G. Dual EGFR/ErbB-2 Inhibitors from Novel Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines. Bioorg. Med. Chem. Lett. 2008, 18 (21), 57385740,  DOI: 10.1016/j.bmcl.2008.09.090
  97. 97
    Stevens, K. L.; Alligood, K. J.; Alberti, J. G. B.; Caferro, T. R.; Chamberlain, S. D.; Dickerson, S. H.; Dickson, H. D.; Emerson, H. K.; Griffin, R. J.; Hubbard, R. D.; Keith, B. R.; Mullin, R. J.; Petrov, K. G.; Gerding, R. M.; Reno, M. J.; Rheault, T. R.; Rusnak, D. W.; Sammond, D. M.; Smith, S. C.; Uehling, D. E.; Waterson, A. G.; Wood, E. R. Synthesis and Stereochemical Effects of Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines as EGFR and ErbB-2 Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (1), 2126,  DOI: 10.1016/j.bmcl.2008.11.023
  98. 98
    Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D. M.; Smith, S. C.; Dickson, H. D.; Caferro, T. R.; Hinkle, K. W.; Stevens, K. L.; Dickerson, S. H.; Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J.; Uehling, D. E. Synthesis and Evaluation of Aniline Headgroups for Alkynyl Thienopyrimidine Dual EGFR/ErbB-2 Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (5), 13321336,  DOI: 10.1016/j.bmcl.2009.01.080
  99. 99
    Nijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R. Design and Pharmacological Characterization of VUF14480, a Covalent Partial Agonist That Interacts with Cysteine 983.36 of the Human Histamine H4 Receptor. Br. J. Pharmacol. 2013, 170, 89100,  DOI: 10.1111/bph.12113
  100. 100
    Schapira, A.; Bate, G.; Kirkpatrick, P. Rasagiline. Nat. Rev. Drug Discovery 2005, 4 (8), 625626,  DOI: 10.1038/nrd1803
  101. 101
    Youdim, M. B. H.; Gross, A.; Finberg, J. P. M. Rasagiline [N-Propargyl-1R(+)-Aminoindan], a Selective and Potent Inhibitor of Mitochondrial Monoamine Oxidase B. Br. J. Pharmacol. 2001, 132 (2), 500506,  DOI: 10.1038/sj.bjp.0703826
  102. 102
    Wright, A. T.; Song, J. D.; Cravatt, B. F. A Suite of Activity-Based Probes for Human Cytochrome P450 Enzymes. J. Am. Chem. Soc. 2009, 131 (30), 1069210700,  DOI: 10.1021/ja9037609
  103. 103
    Wright, A. T.; Cravatt, B. F. Chemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In Vivo. Chem. Biol. 2007, 14 (9), 10431051,  DOI: 10.1016/j.chembiol.2007.08.008
  104. 104
    van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C. Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition. Bioconjugate Chem. 2012, 23 (3), 392398,  DOI: 10.1021/bc200365k
  105. 105
    Tian, H.; Sakmar, T. P.; Huber, T. A Simple Method for Enhancing the Bioorthogonality of Cyclooctyne Reagent. Chem. Commun. 2016, 52 (31), 54515454,  DOI: 10.1039/C6CC01321J
  106. 106
    Haldón, E.; Nicasio, M. C.; Pérez, P. J. Copper-Catalysed Azide–Alkyne Cycloadditions (CuAAC): An Update. Org. Biomol. Chem. 2015, 13 (37), 95289550,  DOI: 10.1039/C5OB01457C
  107. 107
    Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J. R.; Komander, D.; Ovaa, H. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases. J. Am. Chem. Soc. 2013, 135 (8), 28672870,  DOI: 10.1021/ja309802n
  108. 108
    Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Covalent Inhibition of SUMO and Ubiquitin-Specific Cysteine Proteases by an in Situ Thiol–Alkyne Addition. Bioorg. Med. Chem. 2013, 21 (9), 25112517,  DOI: 10.1016/j.bmc.2013.02.039
  109. 109
    Swatek, K. N.; Aumayr, M.; Pruneda, J. N.; Visser, L. J.; Berryman, S.; Kueck, A. F.; Geurink, P. P.; Ovaa, H.; van Kuppeveld, F. J. M.; Tuthill, T. J.; Skern, T.; Komander, D. Irreversible Inactivation of ISG15 by a Viral Leader Protease Enables Alternative Infection Detection Strategies. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), 23712376,  DOI: 10.1073/pnas.1710617115
  110. 110
    Arkona, C.; Rademann, J. Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes. Angew. Chem., Int. Ed. 2013, 52 (32), 82108212,  DOI: 10.1002/anie.201303544
  111. 111
    Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507515,  DOI: 10.1042/bj0390507
  112. 112
    Terrier, F. Rate and Equilibrium Studies in Jackson-Meisenheimer Complexes. Chem. Rev. 1982, 82 (2), 77152,  DOI: 10.1021/cr00048a001
  113. 113
    Terrier, F. Modern Nucleophilic Aromatic Substitution, 1st ed.; Wiley-VCH: Weinheim, 2013.
  114. 114
    Kwan, E. E.; Zeng, Y.; Besser, H. A.; Jacobsen, E. N. Concerted Nucleophilic Aromatic Substitutions. Nat. Chem. 2018, 10 (9), 917923,  DOI: 10.1038/s41557-018-0079-7
  115. 115
    Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.; Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moller, D. E. L-764406 Is a Partial Agonist of Human Peroxisome Proliferator-Activated Receptor γ. The Role of Cys13 in Ligand Binding. J. Biol. Chem. 1999, 274 (12), 79137922,  DOI: 10.1074/jbc.274.12.7913
  116. 116
    Leesnitzer, L. M.; Parks, D. J.; Bledsoe, R. K.; Cobb, J. E.; Collins, J. L.; Consler, T. G.; Davis, R. G.; Hull-Ryde, E. A.; Lenhard, J. M.; Patel, L.; Plunket, K. D.; Shenk, J. L.; Stimmel, J. B.; Therapontos, C.; Willson, T. M.; Blanchard, S. G. Functional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662. Biochemistry 2002, 41 (21), 66406650,  DOI: 10.1021/bi0159581
  117. 117
    Shearer, B. G.; Wiethe, R. W.; Ashe, A.; Billin, A. N.; Way, J. M.; Stanley, T. B.; Wagner, C. D.; Xu, R. X.; Leesnitzer, L. M.; Merrihew, R. V.; Shearer, T. W.; Jeune, M. R.; Ulrich, J. C.; Willson, T. M. Identification and Characterization of 4-Chloro-N-(2-{[5-Trifluoromethyl)-2-Pyridyl]Sulfonyl}ethyl)Benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist. J. Med. Chem. 2010, 53 (4), 18571861,  DOI: 10.1021/jm900464j
  118. 118
    Babaoglu, K.; Simeonov, A.; Irwin, J. J.; Nelson, M. E.; Feng, B.; Thomas, C. J.; Cancian, L.; Costi, M. P.; Maltby, D. A.; Jadhav, A.; Inglese, J.; Austin, C. P.; Shoichet, B. K. Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase. J. Med. Chem. 2008, 51 (8), 25022511,  DOI: 10.1021/jm701500e
  119. 119
    Patterson, J. T.; Asano, S.; Li, X.; Rader, C.; Barbas, C. F. Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone Linkers. Bioconjugate Chem. 2014, 25 (8), 14021407,  DOI: 10.1021/bc500276m
  120. 120
    Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R.; Xian, M. Methylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking Reagent. Org. Lett. 2012, 14 (13), 33963399,  DOI: 10.1021/ol301370s
  121. 121
    Toda, N.; Asano, S.; Barbas, C. F. Rapid, Stable, Chemoselective Labeling of Thiols with Julia–Kocieński-like Reagents: A Serum-Stable Alternative to Maleimide-Based Protein Conjugation. Angew. Chem., Int. Ed. 2013, 52 (48), 1259212596,  DOI: 10.1002/anie.201306241
  122. 122
    Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.; Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135 (16), 59465949,  DOI: 10.1021/ja400119t
  123. 123
    Alapour, S.; de la Torre, B. G.; Ramjugernath, D.; Koorbanally, N. A.; Albericio, F. Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in Bioconjugation. Bioconjugate Chem. 2018, 29 (2), 225233,  DOI: 10.1021/acs.bioconjchem.7b00800
  124. 124
    Brown, S. P.; Smith, A. B. Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/Protein. J. Am. Chem. Soc. 2015, 137 (12), 40344037,  DOI: 10.1021/ja512880g
  125. 125
    Roberts, D. W.; Aptula, A. O. Electrophilic Reactivity and Skin Sensitization Potency of SNAr Electrophiles. Chem. Res. Toxicol. 2014, 27 (2), 240246,  DOI: 10.1021/tx400355n
  126. 126
    Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.; Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.; Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone. J. Biol. Chem. 2011, 286 (14), 1189511908,  DOI: 10.1074/jbc.M110.200436
  127. 127
    Arnold, L. A.; Kosinski, A.; Estébanez-Perpiñá, E.; Guy, R. K. Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators:  Preliminary Structure–Activity Relationships. J. Med. Chem. 2007, 50 (22), 52695280,  DOI: 10.1021/jm070556y
  128. 128
    Visperas, P. R.; Winger, J. A.; Horton, T. M.; Shah, N. H.; Aum, D. J.; Tao, A.; Barros, T.; Yan, Q.; Wilson, C. G.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Modification by Covalent Reaction or Oxidation of Cysteine Residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding. Biochem. J. 2015, 465 (1), 149161,  DOI: 10.1042/BJ20140793
  129. 129
    Visperas, P. R.; Wilson, C. G.; Winger, J. A.; Yan, Q.; Lin, K.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen. SLAS Discov. 2017, 22 (3), 324331,  DOI: 10.1177/1087057116681407
  130. 130
    Shannon, D. A.; Banerjee, R.; Webster, E. R.; Bak, D. W.; Wang, C.; Weerapana, E. Investigating the Proteome Reactivity and Selectivity of Aryl Halides. J. Am. Chem. Soc. 2014, 136 (9), 33303333,  DOI: 10.1021/ja4116204
  131. 131
    Schardon, C. L.; Tuley, A.; Er, J. A. V.; Swartzel, J. C.; Fast, W. Selective Covalent Protein Modification by 4-Halopyridines through Catalysis. ChemBioChem 2017, 18 (15), 15511556,  DOI: 10.1002/cbic.201700104
  132. 132
    Fairhurst, R. A.; Knoepfel, T.; Leblanc, C.; Buschmann, N.; Gaul, C.; Blank, J.; Galuba, I.; Trappe, J.; Zou, C.; Voshol, J.; Genick, C.; Brunet-Lefeuvre, P.; Bitsch, F.; Graus-Porta, D.; Furet, P. Approaches to Selective Fibroblast Growth Factor Receptor 4 Inhibition through Targeting the ATP-Pocket Middle-Hinge Region. MedChemComm 2017, 8, 16041613,  DOI: 10.1039/C7MD00213K
  133. 133
    Hou, W.; Ren, Y.; Zhang, Z.; Sun, H.; Ma, Y.; Yan, B. Novel Quinazoline Derivatives Bearing Various 6-Benzamide Moieties as Highly Selective and Potent EGFR Inhibitors. Bioorg. Med. Chem. 2018, 26 (8), 17401750,  DOI: 10.1016/j.bmc.2018.02.022
  134. 134
    Juchum, M.; Günther, M.; Laufer, S. A. Fighting Cancer Drug Resistance: Opportunities and Challenges for Mutation-Specific EGFR Inhibitors. Drug Resist. Updates 2015, 20, 1228,  DOI: 10.1016/j.drup.2015.05.002
  135. 135
    Chen, K. X.; Lesburg, C. A.; Vibulbhan, B.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennett, F.; Anilkumar, G. N.; Duca, J.; Jiang, Y.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.-C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, F. G. A Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase Inhibitors. J. Med. Chem. 2012, 55 (5), 20892101,  DOI: 10.1021/jm201322r
  136. 136
    Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P. C.; Wang, Z. SAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA Polymerase. J. Med. Chem. 2006, 49 (3), 10341046,  DOI: 10.1021/jm050859x
  137. 137
    Huth, J. R.; Mendoza, R.; Olejniczak, E. T.; Johnson, R. W.; Cothron, D. A.; Liu, Y.; Lerner, C. G.; Chen, J.; Hajduk, P. J. ALARM NMR:  A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens. J. Am. Chem. Soc. 2005, 127 (1), 217224,  DOI: 10.1021/ja0455547
  138. 138
    Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Bioactivation and Hepatotoxicity of Nitroaromatic Drugs. Curr. Drug Metab. 2006, 7, 715727,  DOI: 10.2174/138920006778520606
  139. 139
    Zeng, Q.; Nair, A. G.; Rosenblum, S. B.; Huang, H.-C.; Lesburg, C. A.; Jiang, Y.; Selyutin, O.; Chan, T.-Y.; Bennett, F.; Chen, K. X.; Venkatraman, S.; Sannigrahi, M.; Velazquez, F.; Duca, J. S.; Gavalas, S.; Huang, Y.; Pu, H.; Wang, L.; Pinto, P.; Vibulbhan, B.; Agrawal, S.; Ferrari, E.; Jiang, C.; Li, C.; Hesk, D.; Gesell, J.; Sorota, S.; Shih, N.-Y.; Njoroge, F. G.; Kozlowski, J. A. Discovery of an Irreversible HCV NS5B Polymerase Inhibitor. Bioorg. Med. Chem. Lett. 2013, 23 (24), 65856587,  DOI: 10.1016/j.bmcl.2013.10.060
  140. 140
    Sato, K.; Kunitomo, Y.; Kasai, Y.; Utsumi, S.; Suetake, I.; Tajima, S.; Ichikawa, S.; Matsuda, A. Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-Nucleoside. ChemBioChem 2018, 19 (8), 865872,  DOI: 10.1002/cbic.201700688
  141. 141
    Kasai, Y.; Sato, K.; Utsumi, S.; Ichikawa, S. Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor. ChemBioChem 2018, 19 (17), 18661872,  DOI: 10.1002/cbic.201800244
  142. 142
    Gianatassio, R.; Lopchuk, J. M.; Wang, J.; Pan, C.-M.; Malins, L. R.; Prieto, L.; Brandt, T. A.; Collins, M. R.; Gallego, G. M.; Sach, N. W.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Amination. Science 2016, 351 (6270), 241246,  DOI: 10.1126/science.aad6252
  143. 143
    Lopchuk, J. M.; Fjelbye, K.; Kawamata, Y.; Malins, L. R.; Pan, C.-M.; Gianatassio, R.; Wang, J.; Prieto, L.; Bradow, J.; Brandt, T. A.; Collins, M. R.; Elleraas, J.; Ewanicki, J.; Farrell, W.; Fadeyi, O. O.; Gallego, G. M.; Mousseau, J. J.; Oliver, R.; Sach, N. W.; Smith, J. K.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Heteroatom Functionalization: Development, Scope, and Stereospecificity. J. Am. Chem. Soc. 2017, 139 (8), 32093226,  DOI: 10.1021/jacs.6b13229
  144. 144
    Semmler, K.; Szeimies, G.; Belzner, J. Tetracyclo[5.1.0.01,6.02,7]Octane, a [1.1.1]Propellane Derivative, and a New Route to the Parent Hydrocarbon. J. Am. Chem. Soc. 1985, 107 (22), 64106411,  DOI: 10.1021/ja00308a053
  145. 145
    Haag, B.; Mosrin, M.; Ila, H.; Malakhov, V.; Knochel, P. Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide Bases. Angew. Chem., Int. Ed. 2011, 50 (42), 97949824,  DOI: 10.1002/anie.201101960
  146. 146
    Wishart, D. S.; Feunang, Y. D.; Guo, A. C.; Lo, E. J.; Marcu, A.; Grant, J. R.; Sajed, T.; Johnson, D.; Li, C.; Sayeeda, Z.; Assempour, N.; Iynkkaran, I.; Liu, Y.; Maciejewski, A.; Gale, N.; Wilson, A.; Chin, L.; Cummings, R.; Le, D.; Pon, A.; Knox, C.; Wilson, M. DrugBank 5.0: A Major Update to the DrugBank Database for 2018. Nucleic Acids Res. 2018, 46 (D1), D1074D1082,  DOI: 10.1093/nar/gkx1037
  147. 147
    Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of Fluorine in Medicinal Chemistry. J. Med. Chem. 2015, 58 (21), 83158359,  DOI: 10.1021/acs.jmedchem.5b00258
  148. 148
    Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018, 61 (14), 58225880,  DOI: 10.1021/acs.jmedchem.7b01788
  149. 149
    Grishin, N. V.; Osterman, A. L.; Brooks, H. B.; Phillips, M. A.; Goldsmith, E. J. X-Ray Structure of Ornithine Decarboxylase from Trypanosoma Brucei:  The Native Structure and the Structure in Complex with α-Difluoromethylornithine. Biochemistry 1999, 38 (46), 1517415184,  DOI: 10.1021/bi9915115
  150. 150
    Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2’-Deoxyuridine 5′-Monophosphate. Biochemistry 1994, 33 (50), 1508615094,  DOI: 10.1021/bi00254a018
  151. 151
    Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors. Science 2005, 308 (5726), 13181321,  DOI: 10.1126/science1108367
  152. 152
    Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. A Clickable Inhibitor Reveals Context-Dependent Autoactivation of P90 RSK. Nat. Chem. Biol. 2007, 3 (3), 156160,  DOI: 10.1038/nchembio859
  153. 153
    Carroll, F. A. Perspectives on Structure and Mechanism in Organic Chemistry, 2nd ed.; Wiley-VCH: Weinheim, 2014.
  154. 154
    Shaik, S. S. The .Alpha.- and .Beta.-Carbon Substituent Effect on SN2 Reactivity. A Valence-Bond Approach. J. Am. Chem. Soc. 1983, 105 (13), 43594367,  DOI: 10.1021/ja00351a039
  155. 155
    Lundell, N.; Schreitmüller, T. Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective. Anal. Biochem. 1999, 266 (1), 3147,  DOI: 10.1006/abio.1998.2919
  156. 156
    Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes. Nature 2010, 468 (7325), 790795,  DOI: 10.1038/nature09472
  157. 157
    Weerapana, E.; Simon, G. M.; Cravatt, B. F. Disparate Proteome Reactivity Profiles of Carbon Electrophiles. Nat. Chem. Biol. 2008, 4 (7), 405407,  DOI: 10.1038/nchembio.91
  158. 158
    Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57 (12), 31243137,  DOI: 10.1021/acs.jcim.7b00553
  159. 159
    Allimuthu, D.; Adams, D. J. 2-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe Identification. ACS Chem. Biol. 2017, 12 (8), 21242131,  DOI: 10.1021/acschembio.7b00424
  160. 160
    Steinmetz, C. G.; Xie, P.; Weiner, H.; Hurley, T. D. Structure of Mitochondrial Aldehyde Dehydrogenase: The Genetic Component of Ethanol Aversion. Structure 1997, 5 (5), 701711,  DOI: 10.1016/S0969-2126(97)00224-4
  161. 161
    Karala, A.-R.; Lappi, A.-K.; Ruddock, L. W. Modulation of an Active-Site Cysteine PKa Allows PDI to Act as a Catalyst of Both Disulfide Bond Formation and Isomerization. J. Mol. Biol. 2010, 396 (4), 883892,  DOI: 10.1016/j.jmb.2009.12.014
  162. 162
    Wang, C.; Abegg, D.; Hoch, D. G.; Adibekian, A. Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT. Angew. Chem., Int. Ed. 2016, 55 (8), 29112915,  DOI: 10.1002/anie.201511301
  163. 163
    Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. Ruthenium-Catalyzed Azide–Alkyne Cycloaddition: Scope and Mechanism. J. Am. Chem. Soc. 2008, 130 (28), 89238930,  DOI: 10.1021/ja0749993
  164. 164
    Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide Electrophiles as Activity-Dependent Cysteine Protease Profiling and Discovery Tools. Chem. Biol. 2000, 7 (8), 569581,  DOI: 10.1016/S1074-5521(00)00014-4
  165. 165
    Willems, L. I.; Jiang, J.; Li, K.-Y.; Witte, M. D.; Kallemeijn, W. W.; Beenakker, T. J. N.; Schröder, S. P.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase Probes. Chem. - Eur. J. 2014, 20 (35), 1086410872,  DOI: 10.1002/chem.201404014
  166. 166
    Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P. N -Alkylated Aziridines Are Easily-Prepared, Potent, Specific and Cell-Permeable Covalent Inhibitors of Human β-Glucocerebrosidase. Chem. Commun. 2015, 51 (57), 1139011393,  DOI: 10.1039/C5CC03828F
  167. 167
    Singh, G. S. Synthetic Aziridines in Medicinal Chemistry: A Mini-Review. Mini-Rev. Med. Chem. 2016, 16, 892904,  DOI: 10.2174/1389557515666150709122244
  168. 168
    Pitscheider, M.; Mäusbacher, N.; Sieber, S. A. Antibiotic Activity and Target Discovery of Three-Membered Natural Product-Derived Heterocycles in Pathogenic Bacteria. Chem. Sci. 2012, 3, 20352041,  DOI: 10.1039/c2sc20290e
  169. 169
    Lee, M.; Ikejiri, M.; Klimpel, D.; Toth, M.; Espahbodi, M.; Hesek, D.; Forbes, C.; Kumarasiri, M.; Noll, B. C.; Chang, M.; Mobashery, S. Structure–Activity Relationship for Thiirane-Based Gelatinase Inhibitors. ACS Med. Chem. Lett. 2012, 3 (6), 490495,  DOI: 10.1021/ml300050b
  170. 170
    Harshbarger, W.; Miller, C.; Diedrich, C.; Sacchettini, J. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure 2015, 23 (2), 418424,  DOI: 10.1016/j.str.2014.11.017
  171. 171
    Falagas, M. E.; Vouloumanou, E. K.; Samonis, G.; Vardakas, K. Z. Fosfomycin. Clin. Microbiol. Rev. 2016, 29 (2), 321347,  DOI: 10.1128/CMR.00068-15
  172. 172
    Kim, D. H.; Lees, W. J.; Kempsell, K. E.; Lane, W. S.; Duncan, K.; Walsh, C. T. Characterization of a Cys115 to Asp Substitution in the Escherichia Coli Cell Wall Biosynthetic Enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) That Confers Resistance to Inactivation by the Antibiotic Fosfomycin. Biochemistry 1996, 35 (15), 49234928,  DOI: 10.1021/bi952937w
  173. 173
    Eschenburg, S.; Priestman, M.; Schönbrunn, E. Evidence That the Fosfomycin Target Cys115 in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Is Essential for Product Release. J. Biol. Chem. 2005, 280 (5), 37573763,  DOI: 10.1074/jbc.M411325200
  174. 174
    Porter, N. J.; Christianson, D. W. Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8. ACS Chem. Biol. 2017, 12 (9), 22812286,  DOI: 10.1021/acschembio.7b00330
  175. 175
    Lopus, M.; Smiyun, G.; Miller, H.; Oroudjev, E.; Wilson, L.; Jordan, M. A. Mechanism of Action of Ixabepilone and Its Interactions with the ΒIII-Tubulin Isotype. Cancer Chemother. Pharmacol. 2015, 76 (5), 10131024,  DOI: 10.1007/s00280-015-2863-z
  176. 176
    Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap Portion. J. Med. Chem. 2010, 53 (5), 20382050,  DOI: 10.1021/jm901558p
  177. 177
    Klüter, S.; Simard, J. R.; Rode, H. B.; Grütter, C.; Pawar, V.; Raaijmakers, H. C. A.; Barf, T. A.; Rabiller, M.; van Otterlo, W. A. L.; Rauh, D. Characterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug Resistance. ChemBioChem 2010, 11 (18), 25572566,  DOI: 10.1002/cbic.201000352
  178. 178
    Gehringer, M.; Forster, M.; Laufer, S. A. Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide–Alkyne Cycloaddition. ACS Comb. Sci. 2015, 17 (1), 510,  DOI: 10.1021/co500122h
  179. 179
    Forster, M.; Chaikuad, A.; Bauer, S. M.; Holstein, J.; Robers, M. B.; Corona, C. R.; Gehringer, M.; Pfaffenrot, E.; Ghoreschi, K.; Knapp, S.; Laufer, S. A. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem. Biol. 2016, 23 (11), 13351340,  DOI: 10.1016/j.chembiol.2016.10.008
  180. 180
    Rempel, B. P.; Withers, S. G. Covalent Inhibitors of Glycosidases and Their Applications in Biochemistry and Biology. Glycobiology 2008, 18 (8), 570586,  DOI: 10.1093/glycob/cwn041
  181. 181
    Povirk, L. F.; Shuker, D. E. DNA Damage and Mutagenesis Induced by Nitrogen Mustards. Mutat. Res., Rev. Genet. Toxicol. 1994, 318 (3), 205226,  DOI: 10.1016/0165-1110(94)90015-9
  182. 182
    McGregor, L. M.; Jenkins, M. L.; Kerwin, C.; Burke, J. E.; Shokat, K. M. Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes. Biochemistry 2017, 56 (25), 31783183,  DOI: 10.1021/acs.biochem.7b00271
  183. 183
    Ray, S.; Kreitler, D. F.; Gulick, A. M.; Murkin, A. S. The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition. ACS Chem. Biol. 2018, 13 (6), 14701473,  DOI: 10.1021/acschembio.8b00225
  184. 184
    Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178187,  DOI: 10.1021/bi401432t
  185. 185
    Krenske, E. H.; Petter, R. C.; Houk, K. N. Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to Cysteines. J. Org. Chem. 2016, 81 (23), 1172611733,  DOI: 10.1021/acs.joc.6b02188
  186. 186
    Krishnan, S.; Miller, R. M.; Tian, B.; Mullins, R. D.; Jacobson, M. P.; Taunton, J. Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis. J. Am. Chem. Soc. 2014, 136 (36), 1262412630,  DOI: 10.1021/ja505194w
  187. 187
    Smith, S.; Keul, M.; Engel, J.; Basu, D.; Eppmann, S.; Rauh, D. Characterization of Covalent-Reversible EGFR Inhibitors. ACS Omega 2017, 2 (4), 15631575,  DOI: 10.1021/acsomega.7b00157
  188. 188
    Forster, M.; Chaikuad, A.; Dimitrov, T.; Döring, E.; Holstein, J.; Berger, B.-T.; Gehringer, M.; Ghoreschi, K.; Müller, S.; Knapp, S.; Laufer, S. A. Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]Pyrrolo[2,3-b]Pyridine Scaffold. J. Med. Chem. 2018, 61 (12), 53505366,  DOI: 10.1021/acs.jmedchem.8b00571
  189. 189
    Cheung, S. T.; Miller, M. S.; Pacoma, R.; Roland, J.; Liu, J.; Schumacher, A. M.; Hsieh-Wilson, L. C. Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation. ACS Chem. Biol. 2017, 12 (12), 31263133,  DOI: 10.1021/acschembio.7b00885
  190. 190
    Cully, M. Rational Drug Design: Tuning Kinase Inhibitor Residence Time. Nat. Rev. Drug Discovery 2015, 14, 457,  DOI: 10.1038/nrd4673
  191. 191
    Langrish, C. L.; Bradshaw, J. M.; Owens, T. D.; Campbell, R. L.; Francesco, M. R.; Karr, D. E.; Murray, S. K.; Quesenberry, R. C.; Smith, P. F.; Taylor, M. D.; Zhu, J.; Nunn, P. A.; Gourlay, S. G. PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura. Blood 2017, 130, 1052
  192. 192
    Masjedizadeh, M. R.; Gourlay, S. Salts and Solid Form of a Btk Inhibitor. WO2015127310, August 28, 2015.
  193. 193
    Holm, K. J.; Spencer, C. M. Entacapone. Drugs 1999, 58 (1), 159177,  DOI: 10.2165/00003495-199958010-00017
  194. 194
    Mendgen, T.; Steuer, C.; Klein, C. D. Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal Chemistry. J. Med. Chem. 2012, 55 (2), 743753,  DOI: 10.1021/jm201243p
  195. 195
    Schneider, T. H.; Rieger, M.; Ansorg, K.; Sobolev, A. N.; Schirmeister, T.; Engels, B.; Grabowsky, S. Vinyl Sulfone Building Blocks in Covalently Reversible Reactions with Thiols. New J. Chem. 2015, 39 (7), 58415853,  DOI: 10.1039/C5NJ00368G
  196. 196
    Siklos, M.; BenAissa, M.; Thatcher, G. R. J. Cysteine Proteases as Therapeutic Targets: Does Selectivity Matter? A Systematic Review of Calpain and Cathepsin Inhibitors. Acta Pharm. Sin. B 2015, 5 (6), 506519,  DOI: 10.1016/j.apsb.2015.08.001
  197. 197
    Cleary, J. A.; Doherty, W.; Evans, P.; Malthouse, J. P. G. Quantifying Tetrahedral Adduct Formation and Stabilization in the Cysteine and the Serine Proteases. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 13821391,  DOI: 10.1016/j.bbapap.2015.07.006
  198. 198
    Sanches, M.; Duffy, N. M.; Talukdar, M.; Thevakumaran, N.; Chiovitti, D.; Canny, M. D.; Lee, K.; Kurinov, I.; Uehling, D.; Al-awar, R.; Poda, G.; Prakesch, M.; Wilson, B.; Tam, V.; Schweitzer, C.; Toro, A.; Lucas, J. L.; Vuga, D.; Lehmann, L.; Durocher, D.; Zeng, Q.; Patterson, J. B.; Sicheri, F. Structure and Mechanism of Action of the Hydroxy–Aryl–Aldehyde Class of IRE1 Endoribonuclease Inhibitors. Nat. Commun. 2014, 5, 4202,  DOI: 10.1038/ncomms5202
  199. 199
    Larraufie, M.-H.; Yang, W. S.; Jiang, E.; Thomas, A. G.; Slusher, B. S.; Stockwell, B. R. Incorporation of Metabolically Stable Ketones into a Small Molecule Probe to Increase Potency and Water Solubility. Bioorg. Med. Chem. Lett. 2015, 25 (21), 47874792,  DOI: 10.1016/j.bmcl.2015.07.018
  200. 200
    Cross, B. C. S.; Bond, P. J.; Sadowski, P. G.; Jha, B. K.; Zak, J.; Goodman, J. M.; Silverman, R. H.; Neubert, T. A.; Baxendale, I. R.; Ron, D.; Harding, H. P. The Molecular Basis for Selective Inhibition of Unconventional MRNA Splicing by an IRE1-Binding Small Molecule. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (15), E869E878,  DOI: 10.1073/pnas.1115623109
  201. 201
    Knoepfel, T.; Furet, P.; Mah, R.; Buschmann, N.; Leblanc, C.; Ripoche, S.; Graus-Porta, D.; Wartmann, M.; Galuba, I.; Fairhurst, R. A. 2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4. ACS Med. Chem. Lett. 2018, 9 (3), 215220,  DOI: 10.1021/acsmedchemlett.7b00485
  202. 202
    LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 10811091,  DOI: 10.1021/tx5001046
  203. 203
    Caraballo, R.; Dong, H.; Ribeiro, J. P.; Jiménez-Barbero, J.; Ramström, O. Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System. Angew. Chem., Int. Ed. 2010, 49 (3), 589593,  DOI: 10.1002/anie.200903920
  204. 204
    Buschmann, N.; Fairhurst, R. A.; Knoepfel, T.; Furet, P.; Leblanc, C.; Mah, R.; Kiffe, M.; Graus-Porta, D.; Weiss, A.; Kinyamu-Akunda, J.; Wartmann, M.; Trappe, J.; Gabriel, T. R.; Hofmann, F.; Sellers, W. R. A Reversible Covalent Approach to Selective FGFR4 Inhibition; Book of Abstracts—Frontiers in Medicinal Chemistry Symposium, Jena, 2018; p 26.
  205. 205
    Otto, H.-H.; Schirmeister, T. Cysteine Proteases and Their Inhibitors. Chem. Rev. 1997, 97 (1), 133172,  DOI: 10.1021/cr950025u
  206. 206
    Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. Discovery and Preclinical Profile of Saxagliptin (BMS-477118):  A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes. J. Med. Chem. 2005, 48 (15), 50255037,  DOI: 10.1021/jm050261p
  207. 207
    Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. Nitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile Pharmacophore. J. Med. Chem. 2010, 53 (22), 79027917,  DOI: 10.1021/jm100762r
  208. 208
    Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A Generally Applicable Method for Assessing the Electrophilicity and Reactivity of Diverse Nitrile-Containing Compounds. Bioorg. Med. Chem. Lett. 2007, 17 (4), 9981002,  DOI: 10.1016/j.bmcl.2006.11.044
  209. 209
    Schmitz, J.; Beckmann, A.-M.; Dudic, A.; Li, T.; Sellier, R.; Bartz, U.; Gütschow, M. 3-Cyano-3-Aza-β-Amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins. ACS Med. Chem. Lett. 2014, 5 (10), 10761081,  DOI: 10.1021/ml500238q
  210. 210
    Cai, J.; Fradera, X.; van Zeeland, M.; Dempster, M.; Cameron, K. S.; Bennett, D. J.; Robinson, J.; Popplestone, L.; Baugh, M.; Westwood, P.; Bruin, J.; Hamilton, W.; Kinghorn, E.; Long, C.; Uitdehaag, J. C. M. 4-(3-Trifluoromethylphenyl)-Pyrimidine-2-Carbonitrile as Cathepsin S Inhibitors: N3, Not N1 Is Critically Important. Bioorg. Med. Chem. Lett. 2010, 20 (15), 45074510,  DOI: 10.1016/j.bmcl.2010.06.043
  211. 211
    Mac Sweeney, A.; Grosche, P.; Ellis, D.; Combrink, K.; Erbel, P.; Hughes, N.; Sirockin, F.; Melkko, S.; Bernardi, A.; Ramage, P.; Jarousse, N.; Altmann, E. Discovery and Structure-Based Optimization of Adenain Inhibitors. ACS Med. Chem. Lett. 2014, 5 (8), 937941,  DOI: 10.1021/ml500224t
  212. 212
    de Jesus Cortez, F.; Nguyen, P.; Truillet, C.; Tian, B.; Kuchenbecker, K. M.; Evans, M. J.; Webb, P.; Jacobson, M. P.; Fletterick, R. J.; England, P. M. Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen. ACS Chem. Biol. 2017, 12 (12), 29342939,  DOI: 10.1021/acschembio.7b00702
  213. 213
    Deaton, D. N.; Hassell, A. M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.; Shewchuk, L. M.; Tavares, F. X.; Willard, D. H.; Wright, L. L. Novel and Potent Cyclic Cyanamide-Based Cathepsin K Inhibitors. Bioorg. Med. Chem. Lett. 2005, 15 (7), 18151819,  DOI: 10.1016/j.bmcl.2005.02.033
  214. 214
    Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S. B.; Percival, M. D. Novel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and L. J. Med. Chem. 2001, 44 (1), 94104,  DOI: 10.1021/jm0003440
  215. 215
    Rydzewski, R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S. B.; Bass, K. E.; Wong, D. H. Peptidic 1-Cyanopyrrolidines: Synthesis and SAR of a Series of Potent, Selective Cathepsin Inhibitors. Bioorg. Med. Chem. 2002, 10 (10), 32773284,  DOI: 10.1016/S0968-0896(02)00173-6
  216. 216
    Lainé, D.; Palovich, M.; McCleland, B.; Petitjean, E.; Delhom, I.; Xie, H.; Deng, J.; Lin, G.; Davis, R.; Jolit, A.; Nevins, N.; Zhao, B.; Villa, J.; Schneck, J.; McDevitt, P.; Midgett, R.; Kmett, C.; Umbrecht, S.; Peck, B.; Davis, A. B.; Bettoun, D. Discovery of Novel Cyanamide-Based Inhibitors of Cathepsin C. ACS Med. Chem. Lett. 2011, 2 (2), 142147,  DOI: 10.1021/ml100212k
  217. 217
    Hill, S. V.; Williams, A.; Longridge, J. L. Acid-Catalysed Hydrolysis of Cyanamides: Estimates of Carbodi-Imide Basicity and Tautomeric Equilibrium Constant between Carbodi-Imide and Cyanamide. J. Chem. Soc., Perkin Trans. 2 1984, 0 (6), 10091013,  DOI: 10.1039/p29840001009
  218. 218
    Benson, M. J.; Rodriguez, V.; von Schack, D.; Keegan, S.; Cook, T. A.; Edmonds, J.; Benoit, S.; Seth, N.; Du, S.; Messing, D.; Nickerson-Nutter, C. L.; Dunussi-Joannopoulos, K.; Rankin, A. L.; Ruzek, M.; Schnute, M. E.; Douhan, J. Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune System. J. Immunol. 2014, 193 (1), 185,  DOI: 10.4049/jimmunol.1302570
  219. 219
    Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (1), 173178,  DOI: 10.1073/pnas.1313733111
  220. 220
    Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay. J. Med. Chem. 2012, 55 (22), 1004710063,  DOI: 10.1021/jm301190s
  221. 221
    Gupta, P.; Wright, S. E.; Kim, S.-H.; Srivastava, S. K. Phenethyl Isothiocyanate: A Comprehensive Review of Anti-Cancer Mechanisms. Biochim. Biophys. Acta, Rev. Cancer 2014, 1846 (2), 405424,  DOI: 10.1016/j.bbcan.2014.08.003
  222. 222
    Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. TRP Channel Activation by Reversible Covalent Modification. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (51), 1956419568,  DOI: 10.1073/pnas.0609598103
  223. 223
    van Bladeren, P. J. Glutathione Conjugation as a Bioactivation Reaction. Chem.-Biol. Interact. 2000, 129 (1), 6176,  DOI: 10.1016/S0009-2797(00)00214-3
  224. 224
    Drobnica, L.; Kristián, P.; Augustín, J. The Chemistry of the NCS Group. Cyanates Their Thio Derivatives; Patai, P., Ed.; Wiley: New York, 1977; Part 2, pp 10031221 DOI: 10.1002/9780470771532.ch6 .
  225. 225
    Shibata, T.; Kimura, Y.; Mukai, A.; Mori, H.; Ito, S.; Asaka, Y.; Oe, S.; Tanaka, H.; Takahashi, T.; Uchida, K. Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates. J. Biol. Chem. 2011, 286, 42150,  DOI: 10.1074/jbc.M111.308049
  226. 226
    Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y. Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine. Chem. Res. Toxicol. 2009, 22 (3), 536542,  DOI: 10.1021/tx8003906
  227. 227
    Kumari, V.; Dyba, M. A.; Holland, R. J.; Liang, Y.-H.; Singh, S. V.; Ji, X. Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 2016, 11 (9), e0163821,  DOI: 10.1371/journal.pone.0163821
  228. 228
    Wilson, A. J.; Kerns, J. K.; Callahan, J. F.; Moody, C. J. Keap Calm, and Carry on Covalently. J. Med. Chem. 2013, 56 (19), 74637476,  DOI: 10.1021/jm400224q
  229. 229
    Lewis, S. M.; Li, Y.; Catalano, M. J.; Laciak, A. R.; Singh, H.; Seiner, D. R.; Reilly, T. J.; Tanner, J. J.; Gates, K. S. Inactivation of Protein Tyrosine Phosphatases by Dietary Isothiocyanates. Bioorg. Med. Chem. Lett. 2015, 25 (20), 45494552,  DOI: 10.1016/j.bmcl.2015.08.065
  230. 230
    Cross, J. V.; Foss, F. W.; Rady, J. M.; Macdonald, T. L.; Templeton, D. J. The Isothiocyanate Class of Bioactive Nutrients Covalently Inhibit the MEKK1 Protein Kinase. BMC Cancer 2007, 7 (1), 183,  DOI: 10.1186/1471-2407-7-183
  231. 231
    Mi, L.; Xiao, Z.; Hood, B. L.; Dakshanamurthy, S.; Wang, X.; Govind, S.; Conrads, T. P.; Veenstra, T. D.; Chung, F.-L. Covalent Binding to Tubulin by Isothiocyanates: A Mechanism of Cell Growth Arrest and Apoptosis. J. Biol. Chem. 2008, 283 (32), 2213622146,  DOI: 10.1074/jbc.M802330200
  232. 232
    Ouertatani-Sakouhi, H.; El-Turk, F.; Fauvet, B.; Roger, T.; Le Roy, D.; Karpinar, D. P.; Leng, L.; Bucala, R.; Zweckstetter, M.; Calandra, T.; Lashuel, H. A. A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor. Biochemistry 2009, 48 (41), 98589870,  DOI: 10.1021/bi900957e
  233. 233
    Brown, K. K.; Hampton, M. B. Biological Targets of Isothiocyanates. Biochim. Biophys. Acta, Gen. Subj. 2011, 1810 (9), 888894,  DOI: 10.1016/j.bbagen.2011.06.004
  234. 234
    Pearson, R. J.; Blake, D. G.; Mezna, M.; Fischer, P. M.; Westwood, N. J.; McInnes, C. The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1. Cell Chem. Biol. 2018, 25, 11071116,  DOI: 10.1016/j.chembiol.2018.06.001
  235. 235
    Federici, L.; Lo Sterzo, C.; Pezzola, S.; Di Matteo, A. D.; Scaloni, F.; Federici, G.; Caccuri, A. M. Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases. Cancer Res. 2009, 69 (20), 80258034,  DOI: 10.1158/0008-5472.CAN-09-1314
  236. 236
    Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Site-Directed Ligand Discovery. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (17), 93679372,  DOI: 10.1073/pnas.97.17.9367
  237. 237
    Zong, L.; Bartolami, E.; Abegg, D.; Adibekian, A.; Sakai, N.; Matile, S. Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension. ACS Cent. Sci. 2017, 3 (5), 449453,  DOI: 10.1021/acscentsci.7b00080
  238. 238
    Tjin, C. C.; Otley, K. D.; Baguley, T. D.; Kurup, P.; Xu, J.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases. ACS Cent. Sci. 2017, 3 (12), 13221328,  DOI: 10.1021/acscentsci.7b00486
  239. 239
    Weichert, D.; Gmeiner, P. Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications. ACS Chem. Biol. 2015, 10 (6), 13761386,  DOI: 10.1021/acschembio.5b00070
  240. 240
    Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G. F.; Choi, H.-J.; DeVree, B. T.; Sunahara, R. K.; Chae, P. S.; Gellman, S. H.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Caffrey, M.; Gmeiner, P.; Kobilka, B. K. Structure and Function of an Irreversible Agonist-Β2 Adrenoceptor Complex. Nature 2011, 469 (7329), 236240,  DOI: 10.1038/nature09665
  241. 241
    Schwalbe, T.; Kaindl, J.; Hübner, H.; Gmeiner, P. Potent Haloperidol Derivatives Covalently Binding to the Dopamine D2 Receptor. Bioorg. Med. Chem. 2017, 25 (19), 50845094,  DOI: 10.1016/j.bmc.2017.06.034
  242. 242
    Liu, Y.; Xie, Z.; Zhao, D.; Zhu, J.; Mao, F.; Tang, S.; Xu, H.; Luo, C.; Geng, M.; Huang, M.; Li, J. Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors. J. Med. Chem. 2017, 60 (6), 22272244,  DOI: 10.1021/acs.jmedchem.6b01245
  243. 243
    Napolitano, L.; Scalise, M.; Koyioni, M.; Koutentis, P.; Catto, M.; Eberini, I.; Parravicini, C.; Palazzolo, L.; Pisani, L.; Galluccio, M.; Console, L.; Carotti, A.; Indiveri, C. Potent Inhibitors of Human LAT1 (SLC7A5) Transporter Based on Dithiazole and Dithiazine Compounds for Development of Anticancer Drugs. Biochem. Pharmacol. 2017, 143, 3952,  DOI: 10.1016/j.bcp.2017.07.006
  244. 244
    Nagy, P. Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways. Antioxid. Redox Signaling 2013, 18 (13), 16231641,  DOI: 10.1089/ars.2012.4973
  245. 245
    Go, Y.-M.; Jones, D. P. Thiol/Disulfide Redox States in Signaling and Sensing. Crit. Rev. Biochem. Mol. Biol. 2013, 48 (2), 173181,  DOI: 10.3109/10409238.2013.764840
  246. 246
    García-Santamarina, S.; Boronat, S.; Hidalgo, E. Reversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal Transduction. Biochemistry 2014, 53 (16), 25602580,  DOI: 10.1021/bi401700f
  247. 247
    Gupta, V.; Carroll, K. S. Profiling the Reactivity of Cyclic C-Nucleophiles towards Electrophilic Sulfur in Cysteine Sulfenic Acid. Chem. Sci. 2016, 7 (1), 400415,  DOI: 10.1039/C5SC02569A
  248. 248
    Truong, T. H.; Carroll, K. S. Redox Regulation of Epidermal Growth Factor Receptor Signaling through Cysteine Oxidation. Biochemistry 2012, 51 (50), 99549965,  DOI: 10.1021/bi301441e
  249. 249
    Garcia, F. J.; Carroll, K. S. Redox-Based Probes as Tools to Monitor Oxidized Protein Tyrosine Phosphatases in Living Cells. Eur. J. Med. Chem. 2014, 88, 2833,  DOI: 10.1016/j.ejmech.2014.06.040
  250. 250
    Alcock, L. J.; Farrell, K. D.; Akol, M. T.; Jones, G. H.; Tierney, M. M.; Kramer, H. B.; Pukala, T. L.; Bernardes, G. J. L.; Perkins, M. V.; Chalker, J. M. Norbornene Probes for the Study of Cysteine Oxidation. Tetrahedron 2018, 74 (12), 12201228,  DOI: 10.1016/j.tet.2017.11.011
  251. 251
    Poole, T. H.; Reisz, J. A.; Zhao, W.; Poole, L. B.; Furdui, C. M.; King, S. B. Strained Cycloalkynes as New Protein Sulfenic Acid Traps. J. Am. Chem. Soc. 2014, 136 (17), 61676170,  DOI: 10.1021/ja500364r
  252. 252
    Gupta, V.; Carroll, K. S. Rational Design of Reversible and Irreversible Cysteine Sulfenic Acid-Targeted Linear C-Nucleophiles. Chem. Commun. 2016, 52 (16), 34143417,  DOI: 10.1039/C6CC00228E
  253. 253
    Forman, H. J.; Davies, M. J.; Krämer, A. C.; Miotto, G.; Zaccarin, M.; Zhang, H.; Ursini, F. Protein Cysteine Oxidation in Redox Signaling: Caveats on Sulfenic Acid Detection and Quantification. Arch. Biochem. Biophys. 2017, 617, 2637,  DOI: 10.1016/j.abb.2016.09.013
  254. 254
    Gupta, V.; Yang, J.; Liebler, D. C.; Carroll, K. S. Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles. J. Am. Chem. Soc. 2017, 139 (15), 55885595,  DOI: 10.1021/jacs.7b01791
  255. 255
    Holliday, G. L.; Mitchell, J. B. O.; Thornton, J. M. Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis. J. Mol. Biol. 2009, 390 (3), 560577,  DOI: 10.1016/j.jmb.2009.05.015
  256. 256
    Platzer, G.; Okon, M.; McIntosh, L. P. PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements. J. Biomol. NMR 2014, 60 (2–3), 109129,  DOI: 10.1007/s10858-014-9862-y
  257. 257
    Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; Garcia-Moreno, B. E. Large Shifts in PKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (13), 52605265,  DOI: 10.1073/pnas.1010750108
  258. 258
    Hacker, S. M.; Backus, K. M.; Lazear, M. R.; Forli, S.; Correia, B. E.; Cravatt, B. F. Global Profiling of Lysine Reactivity and Ligandability in the Human Proteome. Nat. Chem. 2017, 9 (12), 11811190,  DOI: 10.1038/nchem.2826
  259. 259
    Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine. Mol. Cell 2000, 6 (4), 909919,  DOI: 10.1016/S1097-2765(05)00089-4
  260. 260
    Wymann, M. P.; Bulgarelli-Leva, G.; Zvelebil, M. J.; Pirola, L.; Vanhaesebroeck, B.; Waterfield, M. D.; Panayotou, G. Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction. Mol. Cell. Biol. 1996, 16 (4), 17221733,  DOI: 10.1128/MCB.16.4.1722
  261. 261
    Pettinger, J.; Le Bihan, Y.-V.; Widya, M.; van Montfort, R. L. M.; Jones, K.; Cheeseman, M. D. An Irreversible Inhibitor of HSP72 That Unexpectedly Targets Lysine-56. Angew. Chem., Int. Ed. 2017, 56 (13), 35363540,  DOI: 10.1002/anie.201611907
  262. 262
    Altmeyer, M.; Amtmann, E.; Heyl, C.; Marschner, A.; Scheidig, A. J.; Klein, C. D. Beta-Aminoketones as Prodrugs for Selective Irreversible Inhibitors of Type-1 Methionine Aminopeptidases. Bioorg. Med. Chem. Lett. 2014, 24 (22), 53105314,  DOI: 10.1016/j.bmcl.2014.09.047
  263. 263
    Dahal, U. P.; Gilbert, A. M.; Obach, R. S.; Flanagan, M. E.; Chen, J. M.; Garcia-Irizarry, C.; Starr, J. T.; Schuff, B.; Uccello, D. P.; Young, J. A. Intrinsic Reactivity Profile of Electrophilic Moieties to Guide Covalent Drug Design: N-α-Acetyl-L-Lysine as an Amine Nucleophile. MedChemComm 2016, 7 (5), 864872,  DOI: 10.1039/C6MD00017G
  264. 264
    Anscombe, E.; Meschini, E.; Mora-Vidal, R.; Martin, M. P.; Staunton, D.; Geitmann, M.; Danielson, U. H.; Stanley, W. A.; Wang, L. Z.; Reuillon, T.; Golding, B. T.; Cano, C.; Newell, D. R.; Noble, M. E. M.; Wedge, S. R.; Endicott, J. A.; Griffin, R. J. Identification and Characterization of an Irreversible Inhibitor of CDK2. Chem. Biol. 2015, 22 (9), 11591164,  DOI: 10.1016/j.chembiol.2015.07.018
  265. 265
    Narayanan, A.; Jones, L. H. Sulfonyl Fluorides as Privileged Warheads in Chemical Biology. Chem. Sci. 2015, 6 (5), 26502659,  DOI: 10.1039/C5SC00408J
  266. 266
    Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry. Angew. Chem., Int. Ed. 2014, 53 (36), 94309448,  DOI: 10.1002/anie.201309399
  267. 267
    Baker, B. R. Irreversible Enzyme Inhibitors. CXLIX. Tissue-Specific Irreversible Inhibitors of Dihydrofolic Reductase. Acc. Chem. Res. 1969, 2 (5), 129136,  DOI: 10.1021/ar50017a001
  268. 268
    Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016, 138 (23), 73537364,  DOI: 10.1021/jacs.6b02960
  269. 269
    Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I. A.; Kelly, J. W. “Inverse Drug Discovery” Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1), 200210,  DOI: 10.1021/jacs.7b08366
  270. 270
    James, G. T. Inactivation of the Protease Inhibitor Phenylmethylsulfonyl Fluoride in Buffers. Anal. Biochem. 1978, 86 (2), 574579,  DOI: 10.1016/0003-2697(78)90784-4
  271. 271
    Lively, M. O.; Powers, J. C. Specificity and Reactivity of Human Granulocyte Elastase and Cathepsin G, Porcine Pancreatic Elastase, Bovine Chymotrypsin and Trypsin toward Inhibition with Sulfonyl Flourides. Biochim. Biophys. Acta BBA - Enzymol. 1978, 525 (1), 171179,  DOI: 10.1016/0005-2744(78)90211-5
  272. 272
    Genov, N. C.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G. Intramolecular Distances between Tryptophan Residues and the Active-Site Serine Residue in Alkaline Bacterial Proteinases as Measured by Fluorescence Energy-Transfer Studies. Biochem. J. 1983, 215 (2), 413416,  DOI: 10.1042/bj2150413
  273. 273
    Esch, F. S.; Allison, W. S. Identification of a Tyrosine Residue at a Nucleotide Binding Site in the Beta Subunit of the Mitochondrial ATPase with P-Fluorosulfonyl[14C]-Benzoyl-5′-Adenosine. J. Biol. Chem. 1978, 253, 61006106
  274. 274
    Jörg, M.; Glukhova, A.; Abdul-Ridha, A.; Vecchio, E. A.; Nguyen, A. T. N.; Sexton, P. M.; White, P. J.; May, L. T.; Christopoulos, A.; Scammells, P. J. Novel Irreversible Agonists Acting at the A1 Adenosine Receptor. J. Med. Chem. 2016, 59 (24), 1118211194,  DOI: 10.1021/acs.jmedchem.6b01561
  275. 275
    Yang, X.; Dong, G.; Michiels, T. J. M.; Lenselink, E. B.; Heitman, L.; Louvel, J.; IJzerman, A. P. A Covalent Antagonist for the Human Adenosine A2A Receptor. Purinergic Signalling 2017, 13 (2), 191201,  DOI: 10.1007/s11302-016-9549-9
  276. 276
    Beauglehole, A. R.; Baker, S. P.; Scammells, P. J. Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine Receptor. J. Med. Chem. 2000, 43 (26), 49734980,  DOI: 10.1021/jm000181f
  277. 277
    Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867877,  DOI: 10.1016/j.cell.2017.01.042
  278. 278
    Kitz, R.; Wilson, I. B. Esters of Methanesulfonic Acid as Irreversible Inhibitors of Acetylcholinesterase. J. Biol. Chem. 1962, 237, 32453249
  279. 279
    Moss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C. Methanesulfonyl Fluoride (MSF): A Double-Blind, Placebo-Controlled Study of Safety and Efficacy in the Treatment of Senile Dementia of the Alzheimer Type. Alzheimer Dis. Assoc. Disord. 1999, 13 (1), 20,  DOI: 10.1097/00002093-199903000-00003
  280. 280
    Moss, D. E.; Fariello, R. G.; Sahlmann, J.; Sumaya, I.; Pericle, F.; Braglia, E. A Randomized Phase I Study of Methanesulfonyl Fluoride, an Irreversible Cholinesterase Inhibitor, for the Treatment of Alzheimer’s Disease. Br. J. Clin. Pharmacol. 2013, 75 (5), 12311239,  DOI: 10.1111/bcp.12018
  281. 281
    Jones, L. H. Reactive Chemical Probes: Beyond the Kinase Cysteinome. Angew. Chem., Int. Ed. 2018, 57 (30), 92209223,  DOI: 10.1002/anie.201802693
  282. 282
    Mukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P. A Study of the Reactivity of S(VI)–F Containing Warheads with Nucleophilic Amino-Acid Side Chains under Physiological Conditions. Org. Biomol. Chem. 2017, 15 (45), 96859695,  DOI: 10.1039/C7OB02028G
  283. 283
    Lundblad, R. L. Chemical Reagents for Protein Modification, 4th ed.; CRC Press: Boca Raton, 2017.
  284. 284
    Chinthakindi, P. K.; Arvidsson, P. I. Sulfonyl Fluorides (SFs): More Than Click Reagents?. Eur. J. Org. Chem. 2018, 2018 (27–28), 36483666,  DOI: 10.1002/ejoc.201800464
  285. 285
    Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.; Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding Fluorosulfate-l-Tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140 (15), 49954999,  DOI: 10.1021/jacs.8b01087
  286. 286
    Zhao, Q.; Ouyang, X.; Wan, X.; Gajiwala, K. S.; Kath, J. C.; Jones, L. H.; Burlingame, A. L.; Taunton, J. Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes. J. Am. Chem. Soc. 2017, 139 (2), 680685,  DOI: 10.1021/jacs.6b08536
  287. 287
    Becher, I.; Savitski, M. M.; Savitski, M. F.; Hopf, C.; Bantscheff, M.; Drewes, G. Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP. ACS Chem. Biol. 2013, 8 (3), 599607,  DOI: 10.1021/cb3005879
  288. 288
    Baranczak, A.; Liu, Y.; Connelly, S.; Du, W.-G. H.; Greiner, E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encalada, S. E.; Kelly, J. W. A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis Elegans. J. Am. Chem. Soc. 2015, 137 (23), 74047414,  DOI: 10.1021/jacs.5b03042
  289. 289
    Grimster, N. P.; Connelly, S.; Baranczak, A.; Dong, J.; Krasnova, L. B.; Sharpless, K. B.; Powers, E. T.; Wilson, I. A.; Kelly, J. W. Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis While Affording a Fluorescent Conjugate. J. Am. Chem. Soc. 2013, 135 (15), 56565668,  DOI: 10.1021/ja311729d
  290. 290
    Dalton, S. E.; Dittus, L.; Thomas, D. A.; Convery, M. A.; Nunes, J.; Bush, J. T.; Evans, J. P.; Werner, T.; Bantscheff, M.; Murphy, J. A.; Campos, S. Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition. J. Am. Chem. Soc. 2018, 140 (3), 932939,  DOI: 10.1021/jacs.7b08979
  291. 291
    Gupta, R. C.; Sachana, M.; Mukherjee, I. M.; Doss, R. B.; Malik, J. K.; Milatovic, D. Organophosphates and Carbamates. In Veterinary Toxicology; Gupta, R. C., Ed.; Academic Press, 2018; pp. 495508,  DOI: 10.1016/B978-0-12-811410-0.00037-4 .
  292. 292
    Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Rapid Labelling and Covalent Inhibition of Intracellular Native Proteins Using Ligand-Directed N -Acyl- N -Alkyl Sulfonamide. Nat. Commun. 2018, 9 (1), 1870,  DOI: 10.1038/s41467-018-04343-0
  293. 293
    Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F. Target Discovery in Small-Molecule Cell-Based Screens by in Situ Proteome Reactivity Profiling. Nat. Biotechnol. 2005, 23, 1303,  DOI: 10.1038/nbt1149
  294. 294
    Evans, M. J.; Morris, G. M.; Wu, J.; Olson, A. J.; Sorensen, E. J.; Cravatt, B. F. Mechanistic and Structural Requirements for Active Site Labeling of Phosphoglycerate Mutase by Spiroepoxides. Mol. BioSyst. 2007, 3, 495506,  DOI: 10.1039/b705113a
  295. 295
    Bongard, J.; Lorenz, M.; Vetter, I. R.; Stege, P.; Porfetye, A. T.; Schmitz, A. L.; Kaschani, F.; Wolf, A.; Koch, U.; Nussbaumer, P.; Klebl, B.; Kaiser, M.; Ehrmann, M. Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent Probes. ACS Chem. Biol. 2018, 13 (5), 13071312,  DOI: 10.1021/acschembio.8b00101
  296. 296
    Diethelm, S.; Schafroth, M. A.; Carreira, E. M. Amine-Selective Bioconjugation Using Arene Diazonium Salts. Org. Lett. 2014, 16 (15), 39083911,  DOI: 10.1021/ol5016509
  297. 297
    Tung, C. L.; Wong, C. T. T.; Fung, E. Y. M.; Li, X. Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification. Org. Lett. 2016, 18 (11), 26002603,  DOI: 10.1021/acs.orglett.6b00983
  298. 298
    Ritter, E.; Przybylski, P.; Brzezinski, B.; Bartl, F. Schiff Bases in Biological Systems. Curr. Org. Chem. 2009, 13 (3), 241249,  DOI: 10.2174/138527209787314805
  299. 299
    Malátková, P.; Wsól, V. Carbonyl Reduction Pathways in Drug Metabolism. Drug Metab. Rev. 2014, 46 (1), 96123,  DOI: 10.3109/03602532.2013.853078
  300. 300
    Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A. V.; Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. Iminoboronates: A New Strategy for Reversible Protein Modification. J. Am. Chem. Soc. 2012, 134 (24), 1029910305,  DOI: 10.1021/ja303436y
  301. 301
    Adams, J.; Kauffman, M. Development of the Proteasome Inhibitor VelcadeTM (Bortezomib). Cancer Invest. 2004, 22 (2), 304311,  DOI: 10.1081/CNV-120030218
  302. 302
    Bandyopadhyay, A.; McCarthy, K. A.; Kelly, M. A.; Gao, J. Targeting Bacteria via Iminoboronate Chemistry of Amine-Presenting Lipids. Nat. Commun. 2015, 6, 6561,  DOI: 10.1038/ncomms7561
  303. 303
    Bandyopadhyay, A.; Gao, J. Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral PH. Chem. - Eur. J. 2015, 21 (42), 1474814752,  DOI: 10.1002/chem.201502077
  304. 304
    Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through Covalent Modification of a Noncatalytic Lysine Side Chain. Nat. Chem. Biol. 2016, 12 (11), 931936,  DOI: 10.1038/nchembio.2174
  305. 305
    Harris, T. K.; Turner, G. J. Structural Basis of Perturbed PKa Values of Catalytic Groups in Enzyme Active Sites. IUBMB Life 2002, 53 (2), 8598,  DOI: 10.1080/15216540211468
  306. 306
    Schwans, J. P.; Sunden, F.; Gonzalez, A.; Tsai, Y.; Herschlag, D. Uncovering the Determinants of a Highly Perturbed Tyrosine PKa in the Active Site of Ketosteroid Isomerase. Biochemistry 2013, 52 (44), 78407855,  DOI: 10.1021/bi401083b
  307. 307
    Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H. Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes. ACS Chem. Biol. 2015, 10 (4), 10941098,  DOI: 10.1021/cb5009475
  308. 308
    Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 1594215943,  DOI: 10.1021/ja0439017
  309. 309
    Jones, M. W.; Mantovani, G.; Blindauer, C. A.; Ryan, S. M.; Wang, X.; Brayden, D. J.; Haddleton, D. M. Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts. J. Am. Chem. Soc. 2012, 134 (17), 74067413,  DOI: 10.1021/ja211855q
  310. 310
    Ban, H.; Gavrilyuk, J.; Barbas, C. F. Tyrosine Bioconjugation through Aqueous Ene-Type Reactions: A Click-Like Reaction for Tyrosine. J. Am. Chem. Soc. 2010, 132 (5), 15231525,  DOI: 10.1021/ja909062q
  311. 311
    Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjugate Chem. 2013, 24 (4), 520532,  DOI: 10.1021/bc300665t
  312. 312
    Hatcher, J. M.; Wu, G.; Zeng, C.; Zhu, J.; Meng, F.; Patel, S.; Wang, W.; Ficarro, S. B.; Leggett, A. L.; Powell, C. E.; Marto, J. A.; Zhang, K.; Ngo, J. C. K.; Fu, X.-D.; Zhang, T.; Gray, N. S. SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform. Cell Chem. Biol. 2018, 25, 460470,  DOI: 10.1016/j.chembiol.2018.01.013
  313. 313
    Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou, D.; Zhang, J.; Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.; Nordin, B.; Weissig, H.; Yang, Q.; Lee, J.-D.; Gray, N. S.; Kozarich, J. W. In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases. Chem. Biol. 2011, 18 (6), 699710,  DOI: 10.1016/j.chembiol.2011.04.011
  314. 314
    Sakamoto, H.; Tsukaguchi, T.; Hiroshima, S.; Kodama, T.; Kobayashi, T.; Fukami, T. A.; Oikawa, N.; Tsukuda, T.; Ishii, N.; Aoki, Y. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell 2011, 19 (5), 679690,  DOI: 10.1016/j.ccr.2011.04.004
  315. 315
    Choi, E. J.; Jung, D.; Kim, J.-S.; Lee, Y.; Kim, B. M. Chemoselective Tyrosine Bioconjugation through Sulfate Click Reaction. Chem. - Eur. J. 2018, 24 (43), 1094810952,  DOI: 10.1002/chem.201802380
  316. 316
    Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.; Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue. ACS Chem. Biol. 2017, 12 (8), 20152020,  DOI: 10.1021/acschembio.7b00403
  317. 317
    Crawford, L. A.; Weerapana, E. A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1). Mol. BioSyst. 2016, 12 (6), 17681771,  DOI: 10.1039/C6MB00250A
  318. 318
    Gehringer, M.; Forster, M.; Pfaffenrot, E.; Bauer, S. M.; Laufer, S. A. Novel Hinge-Binding Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure–Activity Relationship Study on Tofacitinib Bioisosteres. ChemMedChem 2014, 9 (11), 25162527,  DOI: 10.1002/cmdc.201402252
  319. 319
    Gu, C.; Shannon, D. A.; Colby, T.; Wang, Z.; Shabab, M.; Kumari, S.; Villamor, J. G.; McLaughlin, C. J.; Weerapana, E.; Kaiser, M.; Cravatt, B. F.; van der Hoorn, R. A. L. Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases. Chem. Biol. 2013, 20 (4), 541548,  DOI: 10.1016/j.chembiol.2013.01.016
  320. 320
    Ekici, Ö. D.; Paetzel, M.; Dalbey, R. E. Unconventional Serine Proteases: Variations on the Catalytic Ser/His/Asp Triad Configuration. Protein Sci. 2008, 17 (12), 20232037,  DOI: 10.1110/ps.035436.108
  321. 321
    Long, J. Z.; Cravatt, B. F. The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease. Chem. Rev. 2011, 111 (10), 60226063,  DOI: 10.1021/cr200075y
  322. 322
    Lei, J.; Zhou, Y.; Xie, D.; Zhang, Y. Mechanistic Insights into a Classic Wonder Drug—Aspirin. J. Am. Chem. Soc. 2015, 137 (1), 7073,  DOI: 10.1021/ja5112964
  323. 323
    Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What Is in the Name?. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 513,  DOI: 10.1007/s13361-016-1545-3
  324. 324
    Li, Z.; Qian, L.; Li, L.; Bernhammer, J. C.; Huynh, H. V.; Lee, J.-S.; Yao, S. Q. Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications. Angew. Chem., Int. Ed. 2016, 55 (6), 20022006,  DOI: 10.1002/anie.201508104
  325. 325
    Zhao, S.; Dai, J.; Hu, M.; Liu, C.; Meng, R.; Liu, X.; Wang, C.; Luo, T. Photo-Induced Coupling Reactions of Tetrazoles with Carboxylic Acids in Aqueous Solution: Application in Protein Labelling. Chem. Commun. 2016, 52 (25), 47024705,  DOI: 10.1039/C5CC10445A
  326. 326
    Mix, K. A.; Raines, R. T. Optimized Diazo Scaffold for Protein Esterification. Org. Lett. 2015, 17 (10), 23582361,  DOI: 10.1021/acs.orglett.5b00840
  327. 327
    Ban, H. S.; Usui, T.; Nabeyama, W.; Morita, H.; Fukuzawa, K.; Nakamura, H. Discovery of Boron-Conjugated 4-Anilinoquinazoline as a Prolonged Inhibitor of EGFR Tyrosine Kinase. Org. Biomol. Chem. 2009, 7 (21), 44154427,  DOI: 10.1039/b909504g
  328. 328
    Woodward, R. B.; Olofson, R. A.; Mayer, H. A New Synthesis of Peptides. J. Am. Chem. Soc. 1961, 83 (4), 10101012,  DOI: 10.1021/ja01465a072
  329. 329
    Martín-Gago, P.; Fansa, E. K.; Winzker, M.; Murarka, S.; Janning, P.; Schultz-Fademrecht, C.; Baumann, M.; Wittinghofer, A.; Waldmann, H. Covalent Protein Labeling at Glutamic Acids. Cell Chem. Biol. 2017, 24, 589597,  DOI: 10.1016/j.chembiol.2017.03.015
  330. 330
    Martín-Gago, P.; Fansa, E. K.; Klein, C. H.; Murarka, S.; Janning, P.; Schürmann, M.; Metz, M.; Ismail, S.; Schultz-Fademrecht, C.; Baumann, M.; Bastiaens, P. I. H.; Wittinghofer, A.; Waldmann, H. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity That Prevents Efficient Inhibitor Release by Arl2. Angew. Chem., Int. Ed. 2017, 56 (9), 24232428,  DOI: 10.1002/anie.201610957
  331. 331
    Tsukiji, S.; Hamachi, I. Ligand-Directed Tosyl Chemistry for in Situ Native Protein Labeling and Engineering in Living Systems: From Basic Properties to Applications. Curr. Opin. Chem. Biol. 2014, 21, 136143,  DOI: 10.1016/j.cbpa.2014.07.012
  332. 332
    Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Molina, D. M. The Cellular Thermal Shift Assay for Evaluating Drug Target Interactions in Cells. Nat. Protoc. 2014, 9 (9), 21002122,  DOI: 10.1038/nprot.2014.138
  333. 333
    Franken, H.; Mathieson, T.; Childs, D.; Sweetman, G. M. A.; Werner, T.; Tögel, I.; Doce, C.; Gade, S.; Bantscheff, M.; Drewes, G.; Reinhard, F. B. M.; Huber, W.; Savitski, M. M. Thermal Proteome Profiling for Unbiased Identification of Direct and Indirect Drug Targets Using Multiplexed Quantitative Mass Spectrometry. Nat. Protoc. 2015, 10 (10), 15671593,  DOI: 10.1038/nprot.2015.101
  334. 334
    Komissarov, A. A.; Romanova, D. V.; Debabov, V. G. Complete Inactivation of Escherichia Coli Uridine Phosphorylase by Modification of Asp5 with Woodward’s Reagent K. J. Biol. Chem. 1995, 270 (17), 1005010055,  DOI: 10.1074/jbc.270.17.10050
  335. 335
    Qian, Y.; Schürmann, M.; Janning, P.; Hedberg, C.; Waldmann, H. Activity-Based Proteome Profiling Probes Based on Woodward’s Reagent K with Distinct Target Selectivity. Angew. Chem., Int. Ed. 2016, 55 (27), 77667771,  DOI: 10.1002/anie.201602666
  336. 336
    Harlow, K. W.; Switzer, R. L. Chemical Modification of Salmonella Typhimurium Phosphoribosylpyrophosphate Synthetase with 5′-(p-Fluorosulfonylbenzoyl)Adenosine. Identification of an Active Site Histidine. J. Biol. Chem. 1990, 265, 54875493
  337. 337
    Uchida, K.; Stadtman, E. R. Modification of Histidine Residues in Proteins by Reaction with 4-Hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 45444548,  DOI: 10.1073/pnas.89.10.4544
  338. 338
    Yamaguchi, S.; Aldini, G.; Ito, S.; Morishita, N.; Shibata, T.; Vistoli, G.; Carini, M.; Uchida, K. Δ12-Prostaglandin J2 as a Product and Ligand of Human Serum Albumin: Formation of an Unusual Covalent Adduct at His146. J. Am. Chem. Soc. 2010, 132 (2), 824832,  DOI: 10.1021/ja908878n
  339. 339
    Yoshizawa, M.; Itoh, T.; Hori, T.; Kato, A.; Anami, Y.; Yoshimoto, N.; Yamamoto, K. Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands. J. Med. Chem. 2018, 61 (14), 63396349,  DOI: 10.1021/acs.jmedchem.8b00774
  340. 340
    Liu, S.; Widom, J.; Kemp, C. W.; Crews, C. M.; Clardy, J. Structure of Human Methionine Aminopeptidase-2 Complexed with Fumagillin. Science 1998, 282 (5392), 13241327,  DOI: 10.1126/science.282.5392.1324
  341. 341
    Morgen, M.; Jöst, C.; Malz, M.; Janowski, R.; Niessing, D.; Klein, C. D.; Gunkel, N.; Miller, A. K. Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity. ACS Chem. Biol. 2016, 11 (4), 10011011,  DOI: 10.1021/acschembio.5b00755
  342. 342
    Jakob, C. G.; Upadhyay, A. K.; Donner, P. L.; Nicholl, E.; Addo, S. N.; Qiu, W.; Ling, C.; Gopalakrishnan, S. M.; Torrent, M.; Cepa, S. P.; Shanley, J.; Shoemaker, A. R.; Sun, C. C.; Vasudevan, A.; Woller, K. R.; Shotwell, J. B.; Shaw, B.; Bian, Z.; Hutti, J. E. Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315. J. Med. Chem. 2018, 61 (15), 66476657,  DOI: 10.1021/acs.jmedchem.8b00305
  343. 343
    Lin, S.; Yang, X.; Jia, S.; Weeks, A. M.; Hornsby, M.; Lee, P. S.; Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.; Chang, C. J. Redox-Based Reagents for Chemoselective Methionine Bioconjugation. Science 2017, 355 (6325), 597602,  DOI: 10.1126/science.aal3316
  344. 344
    Bizet, V.; Hendriks, C. M. M.; Bolm, C. Sulfur Imidations: Access to Sulfimides and Sulfoximines. Chem. Soc. Rev. 2015, 44 (11), 33783390,  DOI: 10.1039/C5CS00208G
  345. 345
    Gong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A. Releasable and Traceless PEGylation of Arginine-Rich Antimicrobial Peptides. Chem. Sci. 2017, 8 (5), 40824086,  DOI: 10.1039/C7SC00770A
  346. 346
    Seki, Y.; Ishiyama, T.; Sasaki, D.; Abe, J.; Sohma, Y.; Oisaki, K.; Kanai, M. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 2016, 138 (34), 1079810801,  DOI: 10.1021/jacs.6b06692
  347. 347
    Shibata, Y.; Chiba, M. The Role of Extrahepatic Metabolism in the Pharmacokinetics of the Targeted Covalent Inhibitors Afatinib, Ibrutinib, and Neratinib. Drug Metab. Dispos. 2015, 43 (3), 375384,  DOI: 10.1124/dmd.114.061424
  348. 348
    Scheers, E.; Leclercq, L.; de Jong, J.; Bode, N.; Bockx, M.; Laenen, A.; Cuyckens, F.; Skee, D.; Murphy, J.; Sukbuntherng, J.; Mannens, G. Absorption, Metabolism, and Excretion of Oral 14C Radiolabeled Ibrutinib: An Open-Label, Phase I, Single-Dose Study in Healthy Men. Drug Metab. Dispos. 2015, 43, 289297,  DOI: 10.1124/dmd.114.060061
  349. 349
    Chatterjee, P.; Botello-Smith, W. M.; Zhang, H.; Qian, L.; Alsamarah, A.; Kent, D.; Lacroix, J. J.; Baudry, M.; Luo, Y. Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?. J. Am. Chem. Soc. 2017, 139 (49), 1794517952,  DOI: 10.1021/jacs.7b08938
  350. 350
    Alberty, R. A.; Hammes, G. G. Application of the Theory of Diffusion-Controlled Reactions to Enzyme Kinetics. J. Phys. Chem. 1958, 62 (2), 154159,  DOI: 10.1021/j150560a005
  351. 351
    Wright, M. H.; Sieber, S. A. Chemical Proteomics Approaches for Identifying the Cellular Targets of Natural Products. Nat. Prod. Rep. 2016, 33 (5), 681708,  DOI: 10.1039/C6NP00001K

Cited By


This article is cited by 152 publications.

  1. Biplab Keshari Pandia, Chidambaram Gunanathan. Manganese(I) Catalyzed α-Alkenylation of Amides Using Alcohols with Liberation of Hydrogen and Water. The Journal of Organic Chemistry 2021, 86 (15) , 9994-10005. https://doi.org/10.1021/acs.joc.1c00685OpenURL HONG KONG UNIV SCIENCE TECHLGY
  2. Yi Zhang, Deqin Rong, Bingbing Li, Yuanxiang Wang. Targeting Epigenetic Regulators with Covalent Small-Molecule Inhibitors. Journal of Medicinal Chemistry 2021, 64 (12) , 7900-7925. https://doi.org/10.1021/acs.jmedchem.0c02055OpenURL HONG KONG UNIV SCIENCE TECHLGY
  3. Gregory B. Craven, Edward L. Briggs, Charlotte M. Zammit, Alexander McDermott, Stephanie Greed, Dominic P. Affron, Charlotte Leinfellner, Hannah R. Cudmore, Ruth R. Tweedy, Renzo Luisi, James A. Bull, Alan Armstrong. Synthesis and Configurational Assignment of Vinyl Sulfoximines and Sulfonimidamides. The Journal of Organic Chemistry 2021, 86 (11) , 7403-7424. https://doi.org/10.1021/acs.joc.1c00373OpenURL HONG KONG UNIV SCIENCE TECHLGY
  4. Mina Hariri, Fatemeh Darvish, Karen-Pacelye Mengue Me Ndong, Nora Sechet, Geraud Chacktas, Hooriye Boosaliki, Minh Loan Tran Do, Gabin Mwande-Maguene, Jacques Lebibi, Alexander R. Burilov, Tahar Ayad, David Virieux, Jean-Luc Pirat. Gold-Catalyzed Access to Isophosphinoline 2-Oxides. The Journal of Organic Chemistry 2021, 86 (11) , 7813-7824. https://doi.org/10.1021/acs.joc.1c00648OpenURL HONG KONG UNIV SCIENCE TECHLGY
  5. Adrian Hall, Hugues Chanteux, Karelle Ménochet, Marie Ledecq, Monika-Sarah E. D. Schulze. Designing Out PXR Activity on Drug Discovery Projects: A Review of Structure-Based Methods, Empirical and Computational Approaches. Journal of Medicinal Chemistry 2021, 64 (10) , 6413-6522. https://doi.org/10.1021/acs.jmedchem.0c02245OpenURL HONG KONG UNIV SCIENCE TECHLGY
  6. Anthony Mastracchio, Chunqiu Lai, Enrico Digiammarino, Damien B. Ready, Loren M. Lasko, Kenneth D. Bromberg, William J. McClellan, Debra Montgomery, Vlasios Manaves, Bailin Shaw, Mikkel Algire, Melanie J. Patterson, Chaohong C. Sun, Saul Rosenberg, Albert Lai, Michael R. Michaelides. Discovery of a Potent and Selective Covalent p300/CBP Inhibitor. ACS Medicinal Chemistry Letters 2021, 12 (5) , 726-731. https://doi.org/10.1021/acsmedchemlett.0c00654OpenURL HONG KONG UNIV SCIENCE TECHLGY
  7. Hong-Rae Kim, Ravichandra Tagirasa, Euna Yoo. Covalent Small Molecule Immunomodulators Targeting the Protease Active Site. Journal of Medicinal Chemistry 2021, 64 (9) , 5291-5322. https://doi.org/10.1021/acs.jmedchem.1c00172OpenURL HONG KONG UNIV SCIENCE TECHLGY
  8. Elma Mons, Robbert Q. Kim, Bjorn R. van Doodewaerd, Peter A. van Veelen, Monique P. C. Mulder, Huib Ovaa. Exploring the Versatility of the Covalent Thiol–Alkyne Reaction with Substituted Propargyl Warheads: A Deciding Role for the Cysteine Protease. Journal of the American Chemical Society 2021, 143 (17) , 6423-6433. https://doi.org/10.1021/jacs.0c10513OpenURL HONG KONG UNIV SCIENCE TECHLGY
  9. Wuqing Deng, Xiaojuan Chen, Kaili Jiang, Xiaojuan Song, Minhao Huang, Zheng-Chao Tu, Zhang Zhang, Xiaojing Lin, Raquel Ortega, Adam V. Patterson, Jeff B. Smaill, Ke Ding, Suming Chen, Yongheng Chen, Xiaoyun Lu. Investigation of Covalent Warheads in the Design of 2-Aminopyrimidine-based FGFR4 Inhibitors. ACS Medicinal Chemistry Letters 2021, 12 (4) , 647-652. https://doi.org/10.1021/acsmedchemlett.1c00052OpenURL HONG KONG UNIV SCIENCE TECHLGY
  10. Femke A. Meijer, Maxime C. M. van den Oetelaar, Richard G. Doveston, Ella N. R. Sampers, Luc Brunsveld. Covalent Occlusion of the RORγt Ligand Binding Pocket Allows Unambiguous Targeting of an Allosteric Site. ACS Medicinal Chemistry Letters 2021, 12 (4) , 631-639. https://doi.org/10.1021/acsmedchemlett.1c00029OpenURL HONG KONG UNIV SCIENCE TECHLGY
  11. Rambabu N. Reddi, Efrat Resnick, Adi Rogel, Boddu Venkateswara Rao, Ronen Gabizon, Kim Goldenberg, Neta Gurwicz, Daniel Zaidman, Alexander Plotnikov, Haim Barr, Ziv Shulman, Nir London. Tunable Methacrylamides for Covalent Ligand Directed Release Chemistry. Journal of the American Chemical Society 2021, 143 (13) , 4979-4992. https://doi.org/10.1021/jacs.0c10644OpenURL HONG KONG UNIV SCIENCE TECHLGY
  12. Tsuyoshi Ueda, Tomonori Tamura, Masaharu Kawano, Keiya Shiono, Fruzsina Hobor, Andrew J. Wilson, Itaru Hamachi. Enhanced Suppression of a Protein–Protein Interaction in Cells Using Small-Molecule Covalent Inhibitors Based on an N-Acyl-N-alkyl Sulfonamide Warhead. Journal of the American Chemical Society 2021, 143 (12) , 4766-4774. https://doi.org/10.1021/jacs.1c00703OpenURL HONG KONG UNIV SCIENCE TECHLGY
  13. Zhijie Wang, Jiongheng Cai, Jie Cheng, Wenqianzi Yang, Yifan Zhu, Hongmei Li, Tao Lu, Yadong Chen, Shuai Lu. FLT3 Inhibitors in Acute Myeloid Leukemia: Challenges and Recent Developments in Overcoming Resistance. Journal of Medicinal Chemistry 2021, 64 (6) , 2878-2900. https://doi.org/10.1021/acs.jmedchem.0c01851OpenURL HONG KONG UNIV SCIENCE TECHLGY
  14. Ilya V. Nechaev, Georgij V. Cherkaev, Pavel N. Solyev, Nikolay V. Boev. Synthesis and Aerobic Dehydrogenation of Indolizin-1-ol Derivatives. The Journal of Organic Chemistry 2021, 86 (5) , 4220-4235. https://doi.org/10.1021/acs.joc.0c03046OpenURL HONG KONG UNIV SCIENCE TECHLGY
  15. David Huang, Timothy R. Newhouse. Dehydrogenative Pd and Ni Catalysis for Total Synthesis. Accounts of Chemical Research 2021, 54 (5) , 1118-1130. https://doi.org/10.1021/acs.accounts.0c00787OpenURL HONG KONG UNIV SCIENCE TECHLGY
  16. Oliver Laufkötter, Huabin Hu, Filip Miljković, Jürgen Bajorath. Structure- and Similarity-Based Survey of Allosteric Kinase Inhibitors, Activators, and Closely Related Compounds. Journal of Medicinal Chemistry 2021, Article ASAP.OpenURL HONG KONG UNIV SCIENCE TECHLGY
  17. Kerstin Hiesinger, Dmitry Dar’in, Ewgenij Proschak, Mikhail Krasavin. Spirocyclic Scaffolds in Medicinal Chemistry. Journal of Medicinal Chemistry 2021, 64 (1) , 150-183. https://doi.org/10.1021/acs.jmedchem.0c01473OpenURL HONG KONG UNIV SCIENCE TECHLGY
  18. Christian Gampe, Vishal A. Verma. Curse or Cure? A Perspective on the Developability of Aldehydes as Active Pharmaceutical Ingredients. Journal of Medicinal Chemistry 2020, 63 (23) , 14357-14381. https://doi.org/10.1021/acs.jmedchem.0c01177OpenURL HONG KONG UNIV SCIENCE TECHLGY
  19. Paul A. Jackson, Henry A. M. Schares, Katherine F. M. Jones, John C. Widen, Daniel P. Dempe, Francois Grillet, Matthew E. Cuellar, Michael A. Walters, Daniel A. Harki, Kay M. Brummond. Synthesis of Guaianolide Analogues with a Tunable α-Methylene−γ-lactam Electrophile and Correlating Bioactivity with Thiol Reactivity. Journal of Medicinal Chemistry 2020, 63 (23) , 14951-14978. https://doi.org/10.1021/acs.jmedchem.0c01464OpenURL HONG KONG UNIV SCIENCE TECHLGY
  20. Shashank Kulkarni, Klaus Urbahns, Thomas Spangenberg. Targeted Covalent Inhibitors for the Treatment of Malaria?. ACS Infectious Diseases 2020, 6 (11) , 2815-2817. https://doi.org/10.1021/acsinfecdis.0c00684OpenURL HONG KONG UNIV SCIENCE TECHLGY
  21. Robin A. Fairhurst, Thomas Knoepfel, Nicole Buschmann, Catherine Leblanc, Robert Mah, Milen Todorov, Pierre Nimsgern, Sebastien Ripoche, Michel Niklaus, Nicolas Warin, Van Huy Luu, Mario Madoerin, Jasmin Wirth, Diana Graus-Porta, Andreas Weiss, Michael Kiffe, Markus Wartmann, Jacqueline Kinyamu-Akunda, Dario Sterker, Christelle Stamm, Flavia Adler, Alexandra Buhles, Heiko Schadt, Philippe Couttet, Jutta Blank, Inga Galuba, Jörg Trappe, Johannes Voshol, Nils Ostermann, Chao Zou, Jörg Berghausen, Alberto Del Rio Espinola, Wolfgang Jahnke, Pascal Furet. Discovery of Roblitinib (FGF401) as a Reversible-Covalent Inhibitor of the Kinase Activity of Fibroblast Growth Factor Receptor 4. Journal of Medicinal Chemistry 2020, 63 (21) , 12542-12573. https://doi.org/10.1021/acs.jmedchem.0c01019OpenURL HONG KONG UNIV SCIENCE TECHLGY
  22. Zai-Wei Zhang, Shi-Meng Wang, Wan-Yin Fang, Ravindar Lekkala, Hua-Li Qin. Protocol for Stereoselective Construction of Highly Functionalized Dienyl Sulfonyl Fluoride Warheads. The Journal of Organic Chemistry 2020, 85 (21) , 13721-13734. https://doi.org/10.1021/acs.joc.0c01877OpenURL HONG KONG UNIV SCIENCE TECHLGY
  23. Adam Birkholz, David J. Kopecky, Laurie P. Volak, Michael D. Bartberger, Yuping Chen, Christopher M. Tegley, Tara Arvedson, John D. McCarter, Christopher Fotsch, Victor J. Cee. Systematic Study of the Glutathione Reactivity of N-Phenylacrylamides: 2. Effects of Acrylamide Substitution. Journal of Medicinal Chemistry 2020, 63 (20) , 11602-11614. https://doi.org/10.1021/acs.jmedchem.0c00749OpenURL HONG KONG UNIV SCIENCE TECHLGY
  24. Agron Ilazi, Bin Huang, Valery de Almeida Campos, Karl Gademann. Synthesis of Colibactin Pyrrolidono[3,4-d]pyridones via Regioselective C(sp3)–H Activation. Organic Letters 2020, 22 (17) , 6858-6862. https://doi.org/10.1021/acs.orglett.0c02385OpenURL HONG KONG UNIV SCIENCE TECHLGY
  25. Peter Ertl, Eva Altmann, Jeffrey M. McKenna. The Most Common Functional Groups in Bioactive Molecules and How Their Popularity Has Evolved over Time. Journal of Medicinal Chemistry 2020, 63 (15) , 8408-8418. https://doi.org/10.1021/acs.jmedchem.0c00754OpenURL HONG KONG UNIV SCIENCE TECHLGY
  26. Engi Hassaan, Christoph Hohn, Frederik R. Ehrmann, F. Wieland Goetzke, Levon Movsisyan, Tobias Hüfner-Wulsdorf, Maurice Sebastiani, Adrian Härtsch, Klaus Reuter, François Diederich, Gerhard Klebe. Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT. Journal of Medicinal Chemistry 2020, 63 (13) , 6802-6820. https://doi.org/10.1021/acs.jmedchem.0c00115OpenURL HONG KONG UNIV SCIENCE TECHLGY
  27. Fubao Huang, Hangchen Hu, Kai Wang, Chengyuan Peng, Wenwei Xu, Yu Zhang, Jing Gao, Yishen Liu, Hu Zhou, Ruimin Huang, Minjun Li, Jianhua Shen, Yechun Xu. Identification of Highly Selective Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors by a Covalent Fragment-Based Approach. Journal of Medicinal Chemistry 2020, 63 (13) , 7052-7065. https://doi.org/10.1021/acs.jmedchem.0c00372OpenURL HONG KONG UNIV SCIENCE TECHLGY
  28. Ferruccio Palazzesi, Markus R. Hermann, Marc A. Grundl, Alexander Pautsch, Daniel Seeliger, Christofer S. Tautermann, Alexander Weber. BIreactive: A Machine-Learning Model to Estimate Covalent Warhead Reactivity. Journal of Chemical Information and Modeling 2020, 60 (6) , 2915-2923. https://doi.org/10.1021/acs.jcim.9b01058OpenURL HONG KONG UNIV SCIENCE TECHLGY
  29. Tanaji T. Talele. Acetylene Group, Friend or Foe in Medicinal Chemistry. Journal of Medicinal Chemistry 2020, 63 (11) , 5625-5663. https://doi.org/10.1021/acs.jmedchem.9b01617OpenURL HONG KONG UNIV SCIENCE TECHLGY
  30. Kirsten McAulay, Emily A. Hoyt, Morgan Thomas, Marianne Schimpl, Michael S. Bodnarchuk, Hilary J. Lewis, Derek Barratt, Deepa Bhavsar, David M. Robinson, Michael J. Deery, Derek J. Ogg, Gonçalo J. L. Bernardes, Richard A. Ward, Michael J. Waring, Jason G. Kettle. Alkynyl Benzoxazines and Dihydroquinazolines as Cysteine Targeting Covalent Warheads and Their Application in Identification of Selective Irreversible Kinase Inhibitors. Journal of the American Chemical Society 2020, 142 (23) , 10358-10372. https://doi.org/10.1021/jacs.9b13391OpenURL HONG KONG UNIV SCIENCE TECHLGY
  31. Jing Leng, Wenjian Tang, Wan-Yin Fang, Chuang Zhao, Hua-Li Qin. A Simple Protocol for the Stereoselective Construction of Enaminyl Sulfonyl Fluorides. Organic Letters 2020, 22 (11) , 4316-4321. https://doi.org/10.1021/acs.orglett.0c01360OpenURL HONG KONG UNIV SCIENCE TECHLGY
  32. Damien Bosc, Virgyl Camberlein, Ronan Gealageas, Omar Castillo-Aguilera, Benoit Deprez, Rebecca Deprez-Poulain. Kinetic Target-Guided Synthesis: Reaching the Age of Maturity. Journal of Medicinal Chemistry 2020, 63 (8) , 3817-3833. https://doi.org/10.1021/acs.jmedchem.9b01183OpenURL HONG KONG UNIV SCIENCE TECHLGY
  33. Adolfo Cuesta, Xiaobo Wan, Alma L. Burlingame, Jack Taunton. Ligand Conformational Bias Drives Enantioselective Modification of a Surface-Exposed Lysine on Hsp90. Journal of the American Chemical Society 2020, 142 (7) , 3392-3400. https://doi.org/10.1021/jacs.9b09684OpenURL HONG KONG UNIV SCIENCE TECHLGY
  34. Sneha Ray, Andrew S. Murkin. New Electrophiles and Strategies for Mechanism-Based and Targeted Covalent Inhibitor Design. Biochemistry 2019, 58 (52) , 5234-5244. https://doi.org/10.1021/acs.biochem.9b00293OpenURL HONG KONG UNIV SCIENCE TECHLGY
  35. Shengbin Zhou, Juan Pan, Katherine M. Davis, Irene Schaperdoth, Bo Wang, Amie K. Boal, Carsten Krebs, J. Martin Bollinger, Jr.. Steric Enforcement of cis-Epoxide Formation in the Radical C–O-Coupling Reaction by Which (S)-2-Hydroxypropylphosphonate Epoxidase (HppE) Produces Fosfomycin. Journal of the American Chemical Society 2019, 141 (51) , 20397-20406. https://doi.org/10.1021/jacs.9b10974OpenURL HONG KONG UNIV SCIENCE TECHLGY
  36. Lorenzo Cianni, Christian Wolfgang Feldmann, Erik Gilberg, Michael Gütschow, Luiz Juliano, Andrei Leitão, Jürgen Bajorath, Carlos A. Montanari. Can Cysteine Protease Cross-Class Inhibitors Achieve Selectivity?. Journal of Medicinal Chemistry 2019, 62 (23) , 10497-10525. https://doi.org/10.1021/acs.jmedchem.9b00683OpenURL HONG KONG UNIV SCIENCE TECHLGY
  37. Balakrishna Moku, Wan-Yin Fang, Jing Leng, Eric Assen B. Kantchev, Hua-Li Qin. Rh(I)–Diene-Catalyzed Addition of (Hetero)aryl Functionality to 1,3-Dienylsulfonyl Fluorides Achieving Exclusive Regioselectivity and High Enantioselectivity: Generality and Mechanism. ACS Catalysis 2019, 9 (11) , 10477-10488. https://doi.org/10.1021/acscatal.9b03640OpenURL HONG KONG UNIV SCIENCE TECHLGY
  38. Angus Voice, Gary Tresadern, Herman van Vlijmen, Adrian Mulholland. Limitations of Ligand-Only Approaches for Predicting the Reactivity of Covalent Inhibitors. Journal of Chemical Information and Modeling 2019, 59 (10) , 4220-4227. https://doi.org/10.1021/acs.jcim.9b00404OpenURL HONG KONG UNIV SCIENCE TECHLGY
  39. Carlo Baggio, Parima Udompholkul, Luca Gambini, Ahmed F. Salem, Jennifer Jossart, J. Jefferson P. Perry, Maurizio Pellecchia. Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy. Journal of Medicinal Chemistry 2019, 62 (20) , 9188-9200. https://doi.org/10.1021/acs.jmedchem.9b01108OpenURL HONG KONG UNIV SCIENCE TECHLGY
  40. Jessica Plescia, Stéphane De Cesco, Mihai Burai Patrascu, Jerry Kurian, Justin Di Trani, Caroline Dufresne, Alexander S. Wahba, Naëla Janmamode, Anthony K. Mittermaier, Nicolas Moitessier. Integrated Synthetic, Biophysical, and Computational Investigations of Covalent Inhibitors of Prolyl Oligopeptidase and Fibroblast Activation Protein α. Journal of Medicinal Chemistry 2019, 62 (17) , 7874-7884. https://doi.org/10.1021/acs.jmedchem.9b00642OpenURL HONG KONG UNIV SCIENCE TECHLGY
  41. Ferruccio Palazzesi, Marc A. Grundl, Alexander Pautsch, Alexander Weber, Christofer S. Tautermann. A Fast Ab Initio Predictor Tool for Covalent Reactivity Estimation of Acrylamides. Journal of Chemical Information and Modeling 2019, 59 (8) , 3565-3571. https://doi.org/10.1021/acs.jcim.9b00316OpenURL HONG KONG UNIV SCIENCE TECHLGY
  42. Laurent Hoffer, Magali Saez-Ayala, Dragos Horvath, Alexandre Varnek, Xavier Morelli, Philippe Roche. CovaDOTS: In Silico Chemistry-Driven Tool to Design Covalent Inhibitors Using a Linking Strategy. Journal of Chemical Information and Modeling 2019, 59 (4) , 1472-1485. https://doi.org/10.1021/acs.jcim.8b00960OpenURL HONG KONG UNIV SCIENCE TECHLGY
  43. Kangsa Amporndanai, Xiaoli Meng, Weijuan Shang, Zhenmig Jin, Michael Rogers, Yao Zhao, Zihe Rao, Zhi-Jie Liu, Haitao Yang, Leike Zhang, Paul M. O’Neill, S. Samar Hasnain. Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives. Nature Communications 2021, 12 (1) https://doi.org/10.1038/s41467-021-23313-7OpenURL HONG KONG UNIV SCIENCE TECHLGY
  44. Haixia Su, Sheng Yao, Wenfeng Zhao, Yumin Zhang, Jia Liu, Qiang Shao, Qingxing Wang, Minjun Li, Hang Xie, Weijuan Shang, Changqiang Ke, Lu Feng, Xiangrui Jiang, Jingshan Shen, Gengfu Xiao, Hualiang Jiang, Leike Zhang, Yang Ye, Yechun Xu. Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease. Nature Communications 2021, 12 (1) https://doi.org/10.1038/s41467-021-23751-3OpenURL HONG KONG UNIV SCIENCE TECHLGY
  45. Constantin Cretu, Patricia Gee, Xiang Liu, Anant Agrawal, Tuong-Vi Nguyen, Arun K. Ghosh, Andrew Cook, Melissa Jurica, Nicholas A. Larsen, Vladimir Pena. Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors. Nature Communications 2021, 12 (1) https://doi.org/10.1038/s41467-021-24741-1OpenURL HONG KONG UNIV SCIENCE TECHLGY
  46. Xian Zhou, Xuexin Feng, Dachi Wang, Deheng Chen, Gaoxing Wu, Ziqin Yan, Xilin Lyu, Huan Wang, Jin-Ming Yang, Yujun Zhao. Synthesis and bioactivity studies of covalent inhibitors derived from (-)-Chaetominine. Journal of Molecular Structure 2021, 1241 , 130694. https://doi.org/10.1016/j.molstruc.2021.130694OpenURL HONG KONG UNIV SCIENCE TECHLGY
  47. S.A. Adediran, Michael J. Morrison, R.F. Pratt. Detection of an enzyme isomechanism by means of the kinetics of covalent inhibition. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2021, 1869 (9) , 140681. https://doi.org/10.1016/j.bbapap.2021.140681OpenURL HONG KONG UNIV SCIENCE TECHLGY
  48. Wei-hua Wang, Tao Yuan, Mei-jia Qian, Fang-jie Yan, Liu Yang, Qiao-jun He, Bo Yang, Jin-jian Lu, Hong Zhu. Post-translational modification of KRAS: potential targets for cancer therapy. Acta Pharmacologica Sinica 2021, 42 (8) , 1201-1211. https://doi.org/10.1038/s41401-020-00542-yOpenURL HONG KONG UNIV SCIENCE TECHLGY
  49. David Quach, Guanghui Tang, Jothi Anantharajan, Nithya Baburajendran, Anders Poulsen, John L. K. Wee, Priya Retna, Rong Li, Boping Liu, Doris H. Y. Tee, Perlyn Z. Kwek, Joma K. Joy, Wan‐Qi Yang, Chong‐Jing Zhang, Klement Foo, Thomas H. Keller, Shao Q. Yao. Strategic Design of Catalytic Lysine‐Targeting Reversible Covalent BCR‐ABL Inhibitors**. Angewandte Chemie International Edition 2021, 60 (31) , 17131-17137. https://doi.org/10.1002/anie.202105383OpenURL HONG KONG UNIV SCIENCE TECHLGY
  50. David Quach, Guanghui Tang, Jothi Anantharajan, Nithya Baburajendran, Anders Poulsen, John L. K. Wee, Priya Retna, Rong Li, Boping Liu, Doris H. Y. Tee, Perlyn Z. Kwek, Joma K. Joy, Wan‐Qi Yang, Chong‐Jing Zhang, Klement Foo, Thomas H. Keller, Shao Q. Yao. Strategic Design of Catalytic Lysine‐Targeting Reversible Covalent BCR‐ABL Inhibitors**. Angewandte Chemie 2021, 133 (31) , 17268-17274. https://doi.org/10.1002/ange.202105383OpenURL HONG KONG UNIV SCIENCE TECHLGY
  51. Hyunsoo Kim, Yoon Soo Hwang, Mingi Kim, Seung Bum Park. Recent advances in the development of covalent inhibitors. RSC Medicinal Chemistry 2021, 12 (7) , 1037-1045. https://doi.org/10.1039/D1MD00068COpenURL HONG KONG UNIV SCIENCE TECHLGY
  52. Yaniv Tivon, Gianna Falcone, Alexander Deiters. Protein Labeling and Crosslinking by Covalent Aptamers. Angewandte Chemie 2021, 133 (29) , 16035-16040. https://doi.org/10.1002/ange.202101174OpenURL HONG KONG UNIV SCIENCE TECHLGY
  53. Yaniv Tivon, Gianna Falcone, Alexander Deiters. Protein Labeling and Crosslinking by Covalent Aptamers. Angewandte Chemie International Edition 2021, 60 (29) , 15899-15904. https://doi.org/10.1002/anie.202101174OpenURL HONG KONG UNIV SCIENCE TECHLGY
  54. Zhi-Zheng Wang, Xing-Xing Shi, Guang-Yi Huang, Ge-Fei Hao, Guang-Fu Yang. Fragment-based drug design facilitates selective kinase inhibitor discovery. Trends in Pharmacological Sciences 2021, 42 (7) , 551-565. https://doi.org/10.1016/j.tips.2021.04.001OpenURL HONG KONG UNIV SCIENCE TECHLGY
  55. Huabin Hu, Jürgen Bajorath. Systematic assessment of structure-promiscuity relationships between different types of kinase inhibitors. Bioorganic & Medicinal Chemistry 2021, 41 , 116226. https://doi.org/10.1016/j.bmc.2021.116226OpenURL HONG KONG UNIV SCIENCE TECHLGY
  56. Muya Xiong, Haixia Su, Wenfeng Zhao, Hang Xie, Qiang Shao, Yechun Xu. What coronavirus 3C‐like protease tells us: From structure, substrate selectivity, to inhibitor design. Medicinal Research Reviews 2021, 41 (4) , 1965-1998. https://doi.org/10.1002/med.21783OpenURL HONG KONG UNIV SCIENCE TECHLGY
  57. John P. Guilinger, Archna Archna, Martin Augustin, Andreas Bergmann, Paolo A. Centrella, Matthew A. Clark, John W. Cuozzo, Maike Däther, Marie-Aude Guié, Sevan Habeshian, Reiner Kiefersauer, Stephan Krapp, Alfred Lammens, Lukas Lercher, Julie Liu, Yanbin Liu, Klaus Maskos, Michael Mrosek, Klaus Pflügler, Markus Siegert, Heather A. Thomson, Xia Tian, Ying Zhang, Debora L. Konz Makino, Anthony D. Keefe. Novel irreversible covalent BTK inhibitors discovered using DNA-encoded chemistry. Bioorganic & Medicinal Chemistry 2021, 42 , 116223. https://doi.org/10.1016/j.bmc.2021.116223OpenURL HONG KONG UNIV SCIENCE TECHLGY
  58. Yaping Cheng, Jingyuan Wu, Ying Han, Jingyao Xu, Yifan Da, Qian Zhao, Guoying Guo, Yani Zhou, Yimin Chen, Jinghong Liu, Huayao Chen, Xianxing Jiang, Xiaoqing Cai. A CDR-based approach to generate covalent inhibitory antibody for human rhinovirus protease. Bioorganic & Medicinal Chemistry 2021, 42 , 116219. https://doi.org/10.1016/j.bmc.2021.116219OpenURL HONG KONG UNIV SCIENCE TECHLGY
  59. Qingqing Dai, Yuhang Yan, Xiangli Ning, Gen Li, Junlin Yu, Ji Deng, Lingling Yang, Guo-Bo Li. AncPhore: A versatile tool for anchor pharmacophore steered drug discovery with applications in discovery of new inhibitors targeting metallo-β-lactamases and indoleamine/tryptophan 2,3-dioxygenases. Acta Pharmaceutica Sinica B 2021, 11 (7) , 1931-1946. https://doi.org/10.1016/j.apsb.2021.01.018OpenURL HONG KONG UNIV SCIENCE TECHLGY
  60. Jeffrey G. Martin, Jennifer A. Ward, Felix Feyertag, Lu Zhang, Shalise Couvertier, Kevin Guckian, Kilian V. M. Huber, Douglas S. Johnson. Chemoproteomic Profiling of Covalent XPO1 Inhibitors to Assess Target Engagement and Selectivity. ChemBioChem 2021, 22 (12) , 2116-2123. https://doi.org/10.1002/cbic.202100038OpenURL HONG KONG UNIV SCIENCE TECHLGY
  61. Tao Wang, Yanqing Wang, Kai Xu, Yuheng Zhang, Jiarui Guo, Lantao Liu. Transition‐Metal‐Free DMAP‐Mediated Aromatic Esterification of Amides with Organoboronic Acids. European Journal of Organic Chemistry 2021, 2021 (22) , 3274-3277. https://doi.org/10.1002/ejoc.202100478OpenURL HONG KONG UNIV SCIENCE TECHLGY
  62. Yudai Tabuchi, Takahito Watanabe, Riku Katsuki, Yuji Ito, Masumi Taki. Direct screening of a target-specific covalent binder: stringent regulation of warhead reactivity in a matchmaking environment. Chemical Communications 2021, 57 (44) , 5378-5381. https://doi.org/10.1039/D1CC01773JOpenURL HONG KONG UNIV SCIENCE TECHLGY
  63. Matthew D. Lloyd, Maksims Yevglevskis, Amit Nathubhai, Tony D. James, Michael D. Threadgill, Timothy J. Woodman. Racemases and epimerases operating through a 1,1-proton transfer mechanism: reactivity, mechanism and inhibition. Chemical Society Reviews 2021, 50 (10) , 5952-5984. https://doi.org/10.1039/D0CS00540AOpenURL HONG KONG UNIV SCIENCE TECHLGY
  64. Matthias Franz, Britta Mörchen, Carsten Degenhart, Daniel Gülden, Oleksandr Shkura, Dirk Wolters, Uwe Koch, Bert Klebl, Raphael Stoll, Iris Helfrich, Jürgen Scherkenbeck. Sequence‐Selective Covalent CaaX‐Box Receptors Prevent Farnesylation of Oncogenic Ras Proteins and Impact MAPK/PI3 K Signaling. ChemMedChem 2021, 94 https://doi.org/10.1002/cmdc.202100167OpenURL HONG KONG UNIV SCIENCE TECHLGY
  65. Kewei Sun, Zhonghao Sun, Fenglan Zhao, Guangzhi Shan, Qingguo Meng. Recent advances in research of colchicine binding site inhibitors and their interaction modes with tubulin. Future Medicinal Chemistry 2021, 13 (9) , 839-858. https://doi.org/10.4155/fmc-2020-0376OpenURL HONG KONG UNIV SCIENCE TECHLGY
  66. Miljan Kuljanin, Dylan C. Mitchell, Devin K. Schweppe, Ajami S. Gikandi, David P. Nusinow, Nathan J. Bulloch, Ekaterina V. Vinogradova, David L. Wilson, Eric T. Kool, Joseph D. Mancias, Benjamin F. Cravatt, Steven P. Gygi. Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries. Nature Biotechnology 2021, 39 (5) , 630-641. https://doi.org/10.1038/s41587-020-00778-3OpenURL HONG KONG UNIV SCIENCE TECHLGY
  67. Ivy Guan, Kayla Williams, Jolyn Pan, Xuyu Liu. New Cysteine Covalent Modification Strategies Enable Advancement of Proteome‐wide Selectivity of Kinase Modulators. Asian Journal of Organic Chemistry 2021, 10 (5) , 949-963. https://doi.org/10.1002/ajoc.202100036OpenURL HONG KONG UNIV SCIENCE TECHLGY
  68. Julian Breidenbach, Carina Lemke, Thanigaimalai Pillaiyar, Laura Schäkel, Ghazl Al Hamwi, Miriam Diett, Robin Gedschold, Nina Geiger, Vittoria Lopez, Salahuddin Mirza, Vigneshwaran Namasivayam, Anke C. Schiedel, Katharina Sylvester, Dominik Thimm, Christin Vielmuth, Lan Phuong Vu, Maria Zyulina, Jochen Bodem, Michael Gütschow, Christa E. Müller. Die Hauptprotease von SARS‐CoV‐2 als Zielstruktur: Von der Etablierung eines Hochdurchsatz‐Screenings zum Design maßgeschneiderter Inhibitoren. Angewandte Chemie 2021, 133 (18) , 10515-10521. https://doi.org/10.1002/ange.202016961OpenURL HONG KONG UNIV SCIENCE TECHLGY
  69. Julian Breidenbach, Carina Lemke, Thanigaimalai Pillaiyar, Laura Schäkel, Ghazl Al Hamwi, Miriam Diett, Robin Gedschold, Nina Geiger, Vittoria Lopez, Salahuddin Mirza, Vigneshwaran Namasivayam, Anke C. Schiedel, Katharina Sylvester, Dominik Thimm, Christin Vielmuth, Lan Phuong Vu, Maria Zyulina, Jochen Bodem, Michael Gütschow, Christa E. Müller. Targeting the Main Protease of SARS‐CoV‐2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors. Angewandte Chemie International Edition 2021, 60 (18) , 10423-10429. https://doi.org/10.1002/anie.202016961OpenURL HONG KONG UNIV SCIENCE TECHLGY
  70. Kirandeep Samby, Paul A. Willis, Jeremy N. Burrows, Benoît Laleu, Peter J. H. Webborn, . Actives from MMV Open Access Boxes? A suggested way forward. PLOS Pathogens 2021, 17 (4) , e1009384. https://doi.org/10.1371/journal.ppat.1009384OpenURL HONG KONG UNIV SCIENCE TECHLGY
  71. Angus T. Voice, Gary Tresadern, Rebecca M. Twidale, Herman van Vlijmen, Adrian J. Mulholland. Mechanism of covalent binding of ibrutinib to Bruton's tyrosine kinase revealed by QM/MM calculations. Chemical Science 2021, 12 (15) , 5511-5516. https://doi.org/10.1039/D0SC06122KOpenURL HONG KONG UNIV SCIENCE TECHLGY
  72. Clinton G. L. Veale. Into the Fray! A Beginner's Guide to Medicinal Chemistry. ChemMedChem 2021, 16 (8) , 1199-1225. https://doi.org/10.1002/cmdc.202000929OpenURL HONG KONG UNIV SCIENCE TECHLGY
  73. Jiamin Zheng, Jun Wu, Xiao Ding, Hong C. Shen, Ge Zou. Small molecule approaches to treat autoimmune and inflammatory diseases (Part I): Kinase inhibitors. Bioorganic & Medicinal Chemistry Letters 2021, 38 , 127862. https://doi.org/10.1016/j.bmcl.2021.127862OpenURL HONG KONG UNIV SCIENCE TECHLGY
  74. Tasuku Ishida, Alessio Ciulli. E3 Ligase Ligands for PROTACs: How They Were Found and How to Discover New Ones. SLAS DISCOVERY: Advancing the Science of Drug Discovery 2021, 26 (4) , 484-502. https://doi.org/10.1177/2472555220965528OpenURL HONG KONG UNIV SCIENCE TECHLGY
  75. Hong-Ru Chen, Zhen-Yu Hu, Hua-Li Qin, Haolin Tang. A novel three-component reaction for constructing indolizine-containing aliphatic sulfonyl fluorides. Organic Chemistry Frontiers 2021, 8 (6) , 1185-1189. https://doi.org/10.1039/D0QO01430COpenURL HONG KONG UNIV SCIENCE TECHLGY
  76. Thomas A. Baillie. Approaches to mitigate the risk of serious adverse reactions in covalent drug design. Expert Opinion on Drug Discovery 2021, 16 (3) , 275-287. https://doi.org/10.1080/17460441.2021.1832079OpenURL HONG KONG UNIV SCIENCE TECHLGY
  77. Robert Roskoski. Orally effective FDA-approved protein kinase targeted covalent inhibitors (TCIs). Pharmacological Research 2021, 165 , 105422. https://doi.org/10.1016/j.phrs.2021.105422OpenURL HONG KONG UNIV SCIENCE TECHLGY
  78. Valentin Wydra, Stefan Gerstenecker, Dieter Schollmeyer, Stanislav Andreev, Teodor Dimitrov, Ricardo Augusto Massarico Serafim, Stefan Laufer, Matthias Gehringer. N-(6-Chloro-3-nitropyridin-2-yl)-5-(1-methyl-1H-pyrazol-4-yl)isoquinolin-3-amine. Molbank 2021, 2021 (1) , M1181. https://doi.org/10.3390/M1181OpenURL HONG KONG UNIV SCIENCE TECHLGY
  79. Paweł Łukasik, Irena Baranowska-Bosiacka, Katarzyna Kulczycka, Izabela Gutowska. Inhibitors of Cyclin-Dependent Kinases: Types and Their Mechanism of Action. International Journal of Molecular Sciences 2021, 22 (6) , 2806. https://doi.org/10.3390/ijms22062806OpenURL HONG KONG UNIV SCIENCE TECHLGY
  80. Samuel Ofori, Sailajah Gukathasan, Samuel G. Awuah. Gold‐Based Pharmacophore Inhibits Intracellular MYC Protein. Chemistry – A European Journal 2021, 27 (12) , 4168-4175. https://doi.org/10.1002/chem.202004962OpenURL HONG KONG UNIV SCIENCE TECHLGY
  81. Borvornwat Toviwek, Duangkamol Gleeson, M. Paul Gleeson. QM/MM and molecular dynamics investigation of the mechanism of covalent inhibition of TAK1 kinase. Organic & Biomolecular Chemistry 2021, 19 (6) , 1412-1425. https://doi.org/10.1039/D0OB02273JOpenURL HONG KONG UNIV SCIENCE TECHLGY
  82. Rita Fuerst, Rolf Breinbauer. Activity‐Based Protein Profiling (ABPP) of Oxidoreductases. ChemBioChem 2021, 22 (4) , 630-638. https://doi.org/10.1002/cbic.202000542OpenURL HONG KONG UNIV SCIENCE TECHLGY
  83. László Petri, Péter Ábrányi‐Balogh, Imre Tímea, Gyula Pálfy, András Perczel, Damijan Knez, Martina Hrast, Martina Gobec, Izidor Sosič, Kinga Nyíri, Beáta G. Vértessy, Niklas Jänsch, Charlotte Desczyk, Franz‐Josef Meyer‐Almes, Iza Ogris, Simona Golič Grdadolnik, Luca Giacinto Iacovino, Claudia Binda, Stanislav Gobec, György M. Keserű. Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments. ChemBioChem 2021, 22 (4) , 743-753. https://doi.org/10.1002/cbic.202000700OpenURL HONG KONG UNIV SCIENCE TECHLGY
  84. Kemel Arafet, Natalia Serrano-Aparicio, Alessio Lodola, Adrian J. Mulholland, Florenci V. González, Katarzyna Świderek, Vicent Moliner. Mechanism of inhibition of SARS-CoV-2 M pro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity. Chemical Science 2021, 12 (4) , 1433-1444. https://doi.org/10.1039/D0SC06195FOpenURL HONG KONG UNIV SCIENCE TECHLGY
  85. Lei Wang, Louis P. Riel, Bekim Bajrami, Bin Deng, Amy R. Howell, Xudong Yao. α‐Methylene‐β‐Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum. ChemBioChem 2021, 22 (3) , 505-515. https://doi.org/10.1002/cbic.202000605OpenURL HONG KONG UNIV SCIENCE TECHLGY
  86. Sun Dongbang, Jonathan A. Ellman. Synthesis of Nitrile Bearing Acyclic Quaternary Centers through Co(III)‐Catalyzed Sequential C−H Bond Addition to Dienes and N ‐Cyanosuccinimide. Angewandte Chemie International Edition 2021, 60 (4) , 2135-2139. https://doi.org/10.1002/anie.202010735OpenURL HONG KONG UNIV SCIENCE TECHLGY
  87. Sun Dongbang, Jonathan A. Ellman. Synthesis of Nitrile Bearing Acyclic Quaternary Centers through Co(III)‐Catalyzed Sequential C−H Bond Addition to Dienes and N ‐Cyanosuccinimide. Angewandte Chemie 2021, 133 (4) , 2163-2167. https://doi.org/10.1002/ange.202010735OpenURL HONG KONG UNIV SCIENCE TECHLGY
  88. Hongyan Du, Junbo Gao, Gaoqi Weng, Junjie Ding, Xin Chai, Jinping Pang, Yu Kang, Dan Li, Dongsheng Cao, Tingjun Hou. CovalentInDB: a comprehensive database facilitating the discovery of covalent inhibitors. Nucleic Acids Research 2021, 49 (D1) , D1122-D1129. https://doi.org/10.1093/nar/gkaa876OpenURL HONG KONG UNIV SCIENCE TECHLGY
  89. Changyu Huang, Jinpeng Li, Jiaquan Wang, Qingshu Zheng, Zhenhua Li, Tao Tu. Hydrogen-bond-assisted transition-metal-free catalytic transformation of amides to esters. Science China Chemistry 2021, 64 (1) , 66-71. https://doi.org/10.1007/s11426-020-9883-3OpenURL HONG KONG UNIV SCIENCE TECHLGY
  90. Richard A. Ward. Modeling Covalent Protein-Ligand Interactions. 2021,,, 174-189. https://doi.org/10.1016/B978-0-12-801238-3.11519-3OpenURL HONG KONG UNIV SCIENCE TECHLGY
  91. Viktoriya Y. Berdan, Paul C. Klauser, Lei Wang. Covalent peptides and proteins for therapeutics. Bioorganic & Medicinal Chemistry 2021, 29 , 115896. https://doi.org/10.1016/j.bmc.2020.115896OpenURL HONG KONG UNIV SCIENCE TECHLGY
  92. Elena De Vita. 10 years into the resurgence of covalent drugs. Future Medicinal Chemistry 2021, 13 (2) , 193-210. https://doi.org/10.4155/fmc-2020-0236OpenURL HONG KONG UNIV SCIENCE TECHLGY
  93. Solomon Tadesse, Derek R Duckett, Andrii Monastyrskyi. The promise and current status of CDK12/13 inhibition for the treatment of cancer. Future Medicinal Chemistry 2021, 13 (2) , 117-141. https://doi.org/10.4155/fmc-2020-0240OpenURL HONG KONG UNIV SCIENCE TECHLGY
  94. Lyn H. Jones. Target Validation—Prosecuting the Target. 2021,,https://doi.org/10.1016/B978-0-12-820472-6.00014-1OpenURL HONG KONG UNIV SCIENCE TECHLGY
  95. Ernest Awoonor-Williams, Jacob Kennedy, Christopher N. Rowley. Measuring and predicting warhead and residue reactivity. 2021,,, 203-227. https://doi.org/10.1016/bs.armc.2020.09.001OpenURL HONG KONG UNIV SCIENCE TECHLGY
  96. György M. Keserű, Daniel A. Erlanson. The future of covalent inhibition. 2021,,, 267-284. https://doi.org/10.1016/bs.armc.2020.10.003OpenURL HONG KONG UNIV SCIENCE TECHLGY
  97. Leonard Sung. Covalent drugs in development for immune-mediated diseases. 2021,,, 33-74. https://doi.org/10.1016/bs.armc.2021.03.001OpenURL HONG KONG UNIV SCIENCE TECHLGY
  98. Lyn H. Jones. Design of next-generation covalent inhibitors: Targeting residues beyond cysteine. 2021,,, 95-134. https://doi.org/10.1016/bs.armc.2020.10.001OpenURL HONG KONG UNIV SCIENCE TECHLGY
  99. Amit Shraga, Efrat Resnick, Ronen Gabizon, Nir London. Covalent fragment screening. 2021,,, 243-265. https://doi.org/10.1016/bs.armc.2021.04.001OpenURL HONG KONG UNIV SCIENCE TECHLGY
  100. Jianmin Gao, Vincent Nobile. Chemistry perspectives of reversible covalent drugs. 2021,,, 75-94. https://doi.org/10.1016/bs.armc.2020.10.004OpenURL HONG KONG UNIV SCIENCE TECHLGY
Load all citations
  • Abstract

    Figure 1

    Figure 1. Kinetic selectivity of fumaric acid esters. Selectivity is povided by rapid bond formation with the target. Slightly slower ester cleavage deactivates the warhead, preventing even slower labeling of undesired proteins.

    Figure 2

    Figure 2. Ibrutinib-derived fumarate esters and analogous probes equipped with a click handle.

    Figure 3

    Figure 3. Mechanism of cysteine addition to allenamides. The prevalence of the mesomeric structure 9a rationalizes the formation of the nonconjugated product. An alternative mechanism involving attack of the neutral thiol to form a zwitterionic species followed by proton transfer was proposed by Loh and co-workers.

    Figure 4

    Figure 4. Osimertinib-derived allenamides as EGFR inhibitors.

    Figure 5

    Figure 5. Propiolonitriles as potential TCI warheads: (A) 3-Aryl and 3-alkyl propiolonitriles. (B) Mechanism of cysteine addition and thiol exchange.

    Figure 6

    Figure 6. 6-Ethynylthienopyrimidine as covalent ErbB kinase inhibitors. (A) 6-Ethynylthieno[3,2-d]pyrimidine and 6-ethynylthieno[2,3-d]pyrimidine-derived inhibitors. (B) Suggested mechanism of cysteine addition.

    Figure 7

    Figure 7. X-ray crystal structure of compound 17 in complex with the ErbB4 kinase domain (PDB 2R4B). The terminal alkyne moiety has reacted with Cys803 to form a vinyl thioether adduct. The N1-atom of the pyrimidine ring is further anchored to the backbone NH of Met799 in the hinge region via a hydrogen bond. The pyrimidine N3-atom is engaged in a water-mediated hydrogen bond to the side chain of Thr860 preceding the conserved DFG motif.

    Figure 8

    Figure 8. 2-Vinylpyrimidine-derived H4 receptor ligand VUF14480 and the unreactive analogue VUF14481.

    Figure 9

    Figure 9. Nonactivated terminal alkynes as cysteine traps. (A) Reaction of C-terminally propargylated ubiquitin 24 with the active site cysteine in DUBs. (B) Reactive (top) and nonreactive (bottom) analogues.

    Figure 10

    Figure 10. SNAr-based covalent ligands. (A) Classical mechanism of the SNAr reaction with cystein. (B) Selected examples of early SNAr-based ligands and reagents. Leaving groups are highlighted in red.

    Figure 11

    Figure 11. Covalent inhibitors antagonizing the interaction of ZAP-70 and Syk with ITAMs. Leaving groups are highlighted in red.

    Figure 12

    Figure 12. Electron-deficient (hetero)aryl probes used for evaluation of SNAr-based labeling in proteomes. (A) General structures. (B) Selected compounds preferably labeling cysteine or lysine.

    Figure 13

    Figure 13. 4-Halopyridines as quiescent SNAr electrophiles. (A) SNAr-reaction with 4-chloropyridine according to the classical mechanism. An anionic Meisenheimer intermediate is formed. (B) Analogous mechanism of the reaction with N-methyl-4-chloropyridine. A neutral dihydropyridine species is formed as the intermediate. (C) General structure of the investigated compounds. (D) Alkyne-tagged probes used for proteomic analysis.

    Figure 14

    Figure 14. (A) Structure of the covalent FGFR4 inhibitor 41. (B) X-ray cystal structure of 41 in complex with the FGFR4 kinase domain (PDB 5NUD). Both pyridine rings form a hydrogen bond with the backbone NH group of Ala553, while the nitro group stabilizes the active conformation via an intramolecular hydrogen bond with the diarylamino NH. The covalent bond with Cys552 is formed by SNAr displacement of the 6-chloro group from the 2-amino-3-nitropyridine moiety. A weak water mediated H-bond between the nitro substituent and the Arg483 guanidinium group was omitted for clarity.

    Figure 15

    Figure 15. EGFR inhibitors with SNAr warheads which do not form the predicted covalent bond with Cys797.

    Figure 16

    Figure 16. Development of covalent HCV NS5B polymerase inhibitors with SNAr warheads.

    Figure 17

    Figure 17. Second generation NS5B polymerase inhibitors with SNAr warheads. (A) Reversibly binding 2-chloropyridine 49 and the irreversibly binding quinoline analogue 50. (B) X-ray crystal structure of key compound 50 covalently bound to Cys366 of HCV NS5B polymerase (PDB 4MZ4). The compound is further anchored by hydrogen bonds between the 2-pyridone moiety and the backbone carbonyl atom of Gln446 and the NH group of Tyr448. The carboxylate and the quinoline N1-atom are linked to different residues via water-mediated hydrogen bond networks (the second sphere of water molecules and beyond was omitted for clarity). A second conformation of the Cys366 was omitted as well.

    Figure 18

    Figure 18. Strain-release reagents and their reaction with a cysteine-containing peptide.

    Figure 19

    Figure 19. Alkyl halides as CRGs (A) General mechanism of the SN2 reaction. (B) Dual attraction model rationalizing the enhanced reactivity of α-halocarbonyl compounds. (C) Reactivities of α-halopropion- and acetamides in a GSH assay. Half-lives were determined in the presence of 10 mM GSH at pH 7.4 and 37 °Ca or 60 °Cb. N-Phenylacrylamide is shown for comparison. (D) 2-Chloropropionamide (S)-53, a covalent PDIA1 inhibitor.

    Figure 20

    Figure 20. 5-Chloromethyl-1,2,3-triazoles as covalent MGMT inhibitors.

    Figure 21

    Figure 21. Examples of epoxide-containing drugs.

    Figure 22

    Figure 22. α-Acyl epoxides as warheads for putatively covalent EGFR inhibitors.

    Figure 23

    Figure 23. Ruxolitinib-derived triazoles with a propylene oxide warhead as selective JAK3 inhibitors.

    Figure 24

    Figure 24. K-Ras G12D or G12C-targeted covalent inhibitors. Key compound 66a features an aziridine warhead.

    Figure 25

    Figure 25. X-ray crystal structure of the K-Ras G12C mutant covalently bound to compound 66a (PDB 5V6V). Cys12 forms the covalent bond by opening the aziridine ring at the β-position. The indole NH and quinazoline N1-atom are involved in charge-assisted hydrogen bonds to the side chains of of Asp69 and Arg68, respectively, while the piperidine carboxamide oxygen interacts with the side chains of Tyr96 and Asp92 via water-bridged hydrogen bonds.

    Figure 26

    Figure 26. 3-Nitropropionate, propionate-3-nitronate, and the suggested reaction mechanism with ICL.

    Figure 27

    Figure 27. α-Cyanoacrylamide-derived covalent-reversible inhibitors.

    Figure 28

    Figure 28. Optimization of covalent-reversible FGFR4 inhibitors possessing an aldehyde warhead.

    Figure 29

    Figure 29. Reactivities of activated nitriles in a GSH-based assay. Half-lives (increasing from left to right) were determined in the presence of 10 mM GSH at pH 7.4 and 37 °C. Data for common acrylamides are provided for comparison.

    Figure 30

    Figure 30. Bicalutamide-derived antiandrogens with a putative covalent-reversible binding mode. (A) Chemical structures of the compounds studied by England and co-workers. (B) Suggested mechanism of covalent binding to Cys784 of the androgen receptor.

    Figure 31

    Figure 31. PF-303, a covalent-reversible BTK inhibitor featuring a cyanamide warhead.

    Figure 32

    Figure 32. Isothiocyanates as covalent-reversible warheads for cysteine and irreversible CRGs for lysine. (A) Common isothiocyanates found in cruciferous vegetables. (B) Reversible reaction of isothiocyanates with GSH or cysteines in proteins and slow thiourea formation, e.g., with lysine. Possible direct reaction pathways are depicted as dashed arrows.

    Figure 33

    Figure 33. Meisenheimer complex-forming electrophiles as putatively covalent-reversible PLK1 inhibitors. Mechanism and selected compounds.

    Figure 34

    Figure 34. Reversible cysteine targeting by disulfide bond formation. (A) Selected examples of headgroups known to generate disulfide bonds. (B) FAUC50, a covalent ligand which enabled crystallographic structure determination of the β2 adrenergic receptor.

    Figure 35

    Figure 35. Carbon acids adressing cysteine sulfenic acids. (A) Model system used by the Carroll group to assess sulfenic acid labeling with C-nuclophiles. A possible alternative reaction pathway is highlighted by the dashed arrow. (B) Cysteine sulfenic acid formation by ROS and trapping with dimedone. (C) Reactivities of cyclic C-nucleophiles. (D,E) Reactivities of linear C-nucleophiles. (F) Tofacitinib, an approved JAK inhibitor shown to react with sulfenic acids. Reactivity is expressed by pseudo-first-order rate constants derived from an LC-MS assay using the model system shown in (A).

    Figure 36

    Figure 36. Alkyne-tagged C-nucleophilic probes for chemical proteomics studies and their reactivity expressed by second-order rate constants derived from the model system shown in Figure 35A.

    Figure 37

    Figure 37. Probes and warheads used to indentify ligandable lysines by chemical proteomics.

    Figure 38

    Figure 38. Reaction mechanism of wortmannin with Lys833 in PI3Kγ.

    Figure 39

    Figure 39. Reactivity of different Michael acceptors and activated nitriles toward lysine and GSH. Compounds with a preference for GSH are shown the upper row, while such preferably reacting with N-α-acetyl lysine are depicted in the lower row. Half-lives were determined in the presence of 50 mM N-α-acetyl lysine at pH 10.2 and 37 °C or 10 mM GSH at pH 7.4 and 37 °C.

    Figure 40

    Figure 40. Vinyl sulfone targeting a lysine in the solvent-exposed front region of CDK2. (A) Covalent and noncovalent inhibitors. (B) X-ray crystal structure of vinyl sulfone 98a covalently bound to Lys89 flanking the solvent-exposed front region of CDK2 (PDB 5CYI). The purine NH is hydrogen-bonded to the backbone carbonyl atom of Glu81, while the N3-atom and the diaryl NH are anchored to the backbone of Leu83 by two additional hydrogen bonds. Another water-bridged hydrogen bond links the purine N7-atom to the backbone NH group of Asp145 in the DFG motif. Further direct and water-mediated hydrogen bonds are established by the sulfonyl group. The N-terminal lobe was omitted for clarity.

    Figure 41

    Figure 41. Sulfur (VI) fluorides for lysine and tyrosine targeting.

    Figure 42

    Figure 42. Examples of sulfonyl fluorides applied in medicinal chemistry and chemical biology.

    Figure 43

    Figure 43. Compounds used for assessing the reactivity of sulfur (VI) fluorides toward different amino acids. (A) sulfonyl fluorides. (B) sulfonimidoyl fluorides. (C) aryl fluorosulfates. (D) Mechanism of the reaction between phenylsulfonyl fluoride and NAC.

    Figure 44

    Figure 44. Lysine-targeted m-5′-FSBA analogues for the evaluation of the relationship between warhead reactivity and FGFR1 inhibitory activity.

    Figure 45

    Figure 45. (A) Promiscuous kinase probe XO44. (B) X-ray crystal structure of XO44 covalently bound to the conserved Lys295 in the kinase SRC (PDB 5K9I). The 3-aminopyrazole is anchored to the hinge region by three hydrogen bonds involving the backbone of Glu339 and Met341. The sulfonyl group forms two additional hydrogen bonds with Phe278 and Gly279 in the glycine-rich loop, while the propargyl amide tag is oriented toward the bulk solvent without being involved in specific interactions.

    Figure 46

    Figure 46. Fluorosulfates for lysine targeting. (A) Aryl fluorosulfates and sulfonyl fluorides designed for adressing Lys15 in human transthyretin. (B) X-ray crystal structure of 113b bound to human transthyretin (PDB 4YDM). Unexpectedly, the free Lys15 ε-sulfamate was observed instead of the covalently bound ligand. The ligand is located in a relatively shallow pocket on the protein surface forming only a single conserved hydrogen bond between the hydroxy group of the dichlorophenol moiety and the side chain of Ser117. An alternative orientation of the 3-hydroxyphenyl residue was omitted for clarity.

    Figure 47

    Figure 47. Development of activated esters covalently targeting Lys779 in PI3Kδ.

    Figure 48

    Figure 48. N-Acyl-N-alkyl sulfonamides addressing surface-exposed lysine side chains. (A) Biotin-transferring probes. (B) Covalent ligand design and application example. The transferable residue is highlighted in red.

    Figure 49

    Figure 49. 2-Formylbenzenboronic acid reversibly forming stabilized Schiff bases with amines.

    Figure 50

    Figure 50. 2-Formylbenzenboronic acids and analogous acetophenones targeting MCL-1 via Schiff base formation.

    Figure 51

    Figure 51. Design of tyrosine-targeted sulfonyl fluorides as DcpS inhibitors.

    Figure 52

    Figure 52. Distinct binding modes of 124ac in the respective X-ray crystal structures in complex with DcpS. The ligand is completely embedded in the protein environment, and the 2,4-diaminquinazoline core adopts a similar orientation in all structures. Conserved interactions include two hydrogen bonds of the quinazoline 2-amino group to the carboxylate of Glu185 and the backbone carbonyl of Pro204 as well as a hydrogen bond between the quinazoline 4-amino group and the Asp205 side chain. The second proton of the quinazoline 4-amine forms an intramolecular H-bond to the ether linker. The (protonated) quinazoline N1-atom is also hydrogen-bonded to Glu185. (A) ortho-Substituted derivative 124a covalently bound to Tyr113 (PDB 4QDE). The phenyl ring points to the “bottom” of the binding pocket. Additional hydrogen bonds are formed to the side chains of Lys142 and Tyr273. (B) meta-Substituted derivative 124b covalently bound to Tyr113 (PDB 4QEB). Covalent attachment is enabled by an “upward” orientation of the phenyl ring, giving rise to an additional hydrogen bond between the sulfonyl group and the Tyr143 side chain. (C) para-Substituted derivative 124c covalently bound to Tyr143 (PDB 4QDV). The overall orientation resembles that of 124a, but Tyr143 is labeled instead of Tyr113. No hydrogen bonds with Lys142, Tyr273 and His139 are observed in (B) and (C), and the latter two residues were omitted for clarity. No covalent modification of the proximal nucleophiles Lys142 and His139 was observed in any of the experiments.

    Figure 53

    Figure 53. Design of sulfonyl fluoride SRPKIN-1, the first tyrosine-targeted covalent kinase inhibitor.

    Figure 54

    Figure 54. Aryl fluorosulfate probes targeting CRABP2 used in chemical proteomics studies.

    Figure 55

    Figure 55. X-ray crystal structure of the fluorosulfate-based ligand 128 covalently bound to Tyr134 in CRABP2 (PDB 5HZQ). The ligand is deeply buried in the binding site, and the sulfate group is engaged in a direct hydrogen bond to the Arg132 side chain and water-mediated hydrogen bonds to the Arg111 side chain. The PEG-linker is not resolved and a second, slightly deviating conformation of the ligand and the Tyr134 side chain was omitted for clarity.

    Figure 56

    Figure 56. Alkyne-tagged aryl fluorosulfate-based probes used in an “inverse drug discovery” approach.

    Figure 57

    Figure 57. LAS17, a tyrosine-targeted dichlorotriazine-derived GSTP1 inhibitor.

    Figure 58

    Figure 58. (A) Aryl fluorosulfate-based inhibitor FS-p1 targeting Ser272 in DcpS. (B) Proposed mechanism for the formation of the dehydroalanine elimination product.

    Figure 59

    Figure 59. Glutamate-targeted PDE6δ inhibitors. (A) Attachment of N-methyl isoxazolium warheads to reversible inhibitors exemplified by 135. (B) Mechanism of the reaction between the N-methyl isoxazolium group and carboxylates.

    Figure 60

    Figure 60. X-ray crystal structure of compound 140d covalently bound to Glu88 of PDE6δ (PDB 5NAL). The ligand is predominantly bound in the less stable O-acylated form and deeply buried in the binding site. Hydrogen bonds are formed by both oxygen atoms of the first sulfonyl group to the side chains of Arg61 and Gln78. An additional hydrogen bond is established between the second sulfonyl group and the side chain of Tyr149 (omitted for clarity).

    Figure 61

    Figure 61. Spiro-epoxides as histidine-targeted covalent inhibitors of hMetAP2. (A) Former drug candidate beloranib. (B) Reaction of the natural product fumagillin with His231 in hMetAP2. (C) Simplified fumagillin-derived structures. (D) hMetAP2 inhibitor 145a with improved PK properties.

    Figure 62

    Figure 62. X-ray crystal structure of the covalent complex between hMetAP2 and compound 145a (PDB 5CLS). His231 is covalently attached to the methylene group formed from the terminal carbon atom of the epoxide ring. The ensuing hydroxy group is linked to Asp251 and His382 by water-mediated hydrogen bonds. A direct hydrogen bond between the carbamate’s carbonyl group and the Asn329 backbone NH, and further water-mediated hydrogen bonds additionally anchor the ligand in the binding site.

    Figure 63

    Figure 63. α-Cyanoenones as histidine-targeted covalent-reversible IDH1 inhibitors. (A) Hit compound 146a and optimized derivatives. (B) X-ray crystal structure of 146c bound to IDH1 (PDB 6BL1). His315 is covalently attached to the β-position of the enone precursor. A key hydrogen bond is formed between the α-cyano moiety and the backbone NH of Se326. The enone keto group is hydrogen-bonded to the Lys374 side chain, while the diarylamino group forms a charge-assisted hydrogen bond to the carboxylate of Asp375.

    Figure 64

    Figure 64. Methionine-targeted oxaziridines. (A) Urea and carbamate-derived analogues. (B) Reaction with methionine via S-imidation or concomitant S-oxidation. Conditions: (1) D2O/CD3OD = 1:1, 2.5 min or (2) D2O/CD3OD = 95:5, 20 min. (C) Reaction mechanism of covalent methionine modification.

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 351 other publications.

    1. 1
      Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nat. Biotechnol. 2003, 21 (3), 255261,  DOI: 10.1038/nbt0303-255
    2. 2
      Schopfer, F. J.; Cipollina, C.; Freeman, B. A. Formation and Signaling Actions of Electrophilic Lipids. Chem. Rev. 2011, 111 (10), 59976021,  DOI: 10.1021/cr200131e
    3. 3
      Allis, C. D.; Jenuwein, T. The Molecular Hallmarks of Epigenetic Control. Nat. Rev. Genet. 2016, 17 (8), 487500,  DOI: 10.1038/nrg.2016.59
    4. 4
      Uetrecht, J. Idiosyncratic Drug Reactions: Current Understanding. Annu. Rev. Pharmacol. Toxicol. 2007, 47 (1), 513539,  DOI: 10.1146/annurev.pharmtox.47.120505.105150
    5. 5
      Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. The Resurgence of Covalent Drugs. Nat. Rev. Drug Discovery 2011, 10 (4), 307317,  DOI: 10.1038/nrd3410
    6. 6
      Bauer, R. A. Covalent Inhibitors in Drug Discovery: From Accidental Discoveries to Avoided Liabilities and Designed Therapies. Drug Discovery Today 2015, 20 (9), 10611073,  DOI: 10.1016/j.drudis.2015.05.005
    7. 7
      Powers, J. C.; Asgian, J. L.; Ekici, Ö. D.; James, K. E. Irreversible Inhibitors of Serine, Cysteine, and Threonine Proteases. Chem. Rev. 2002, 102 (12), 46394750,  DOI: 10.1021/cr010182v
    8. 8
      Bachovchin, D. A.; Cravatt, B. F. The Pharmacological Landscape and Therapeutic Potential of Serine Hydrolases. Nat. Rev. Drug Discovery 2012, 11 (1), 5268,  DOI: 10.1038/nrd3620
    9. 9
      Paul, S. M.; Mytelka, D. S.; Dunwiddie, C. T.; Persinger, C. C.; Munos, B. H.; Lindborg, S. R.; Schacht, A. L. How to Improve R&D Productivity: The Pharmaceutical Industry’s Grand Challenge. Nat. Rev. Drug Discovery 2010, 9 (3), 203214,  DOI: 10.1038/nrd3078
    10. 10
      Bandyopadhyay, A.; Gao, J. Targeting Biomolecules with Reversible Covalent Chemistry. Curr. Opin. Chem. Biol. 2016, 34, 110116,  DOI: 10.1016/j.cbpa.2016.08.011
    11. 11
      Bradshaw, J. M.; McFarland, J. M.; Paavilainen, V. O.; Bisconte, A.; Tam, D.; Phan, V. T.; Romanov, S.; Finkle, D.; Shu, J.; Patel, V.; Ton, T.; Li, X.; Loughhead, D. G.; Nunn, P. A.; Karr, D. E.; Gerritsen, M. E.; Funk, J. O.; Owens, T. D.; Verner, E.; Brameld, K. A.; Hill, R. J.; Goldstein, D. M.; Taunton, J. Prolonged and Tunable Residence Time Using Reversible Covalent Kinase Inhibitors. Nat. Chem. Biol. 2015, 11 (7), 525531,  DOI: 10.1038/nchembio.1817
    12. 12
      Flanagan, M. E.; Abramite, J. A.; Anderson, D. P.; Aulabaugh, A.; Dahal, U. P.; Gilbert, A. M.; Li, C.; Montgomery, J.; Oppenheimer, S. R.; Ryder, T.; Schuff, B. P.; Uccello, D. P.; Walker, G. S.; Wu, Y.; Brown, M. F.; Chen, J. M.; Hayward, M. M.; Noe, M. C.; Obach, R. S.; Philippe, L.; Shanmugasundaram, V.; Shapiro, M. J.; Starr, J.; Stroh, J.; Che, Y. Chemical and Computational Methods for the Characterization of Covalent Reactive Groups for the Prospective Design of Irreversible Inhibitors. J. Med. Chem. 2014, 57 (23), 1007210079,  DOI: 10.1021/jm501412a
    13. 13
      Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.; Horning, B. D.; González-Páez, G. E.; Chatterjee, S.; Lanning, B. R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F. Proteome-Wide Covalent Ligand Discovery in Native Biological Systems. Nature 2016, 534 (7608), 570574,  DOI: 10.1038/nature18002
    14. 14
      Miller, R. M.; Paavilainen, V. O.; Krishnan, S.; Serafimova, I. M.; Taunton, J. Electrophilic Fragment-Based Design of Reversible Covalent Kinase Inhibitors. J. Am. Chem. Soc. 2013, 135 (14), 52985301,  DOI: 10.1021/ja401221b
    15. 15
      Jöst, C.; Nitsche, C.; Scholz, T.; Roux, L.; Klein, C. D. Promiscuity and Selectivity in Covalent Enzyme Inhibition: A Systematic Study of Electrophilic Fragments. J. Med. Chem. 2014, 57 (18), 75907599,  DOI: 10.1021/jm5006918
    16. 16
      Kathman, S. G.; Xu, Z.; Statsyuk, A. V. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases. J. Med. Chem. 2014, 57 (11), 49694974,  DOI: 10.1021/jm500345q
    17. 17
      Ostrem, J. M.; Peters, U.; Sos, M. L.; Wells, J. A.; Shokat, K. M. K-Ras(G12C) Inhibitors Allosterically Control GTP Affinity and Effector Interactions. Nature 2013, 503 (7477), 548551,  DOI: 10.1038/nature12796
    18. 18
      Zimmermann, G.; Rieder, U.; Bajic, D.; Vanetti, S.; Chaikuad, A.; Knapp, S.; Scheuermann, J.; Mattarella, M.; Neri, D. A Specific and Covalent JNK-1 Ligand Selected from an Encoded Self-Assembling Chemical Library. Chem. - Eur. J. 2017, 23 (34), 81528155,  DOI: 10.1002/chem.201701644
    19. 19
      Zambaldo, C.; Daguer, J.-P.; Saarbach, J.; Barluenga, S.; Winssinger, N. Screening for Covalent Inhibitors Using DNA-Display of Small Molecule Libraries Functionalized with Cysteine Reactive Moieties. MedChemComm 2016, 7 (7), 13401351,  DOI: 10.1039/C6MD00242K
    20. 20
      Strelow, J. M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition. SLAS Discov. 2017, 22 (1), 320,  DOI: 10.1177/1087057116671509
    21. 21
      Miyahisa, I.; Sameshima, T.; Hixon, M. S. Rapid Determination of the Specificity Constant of Irreversible Inhibitors (Kinact/Ki) by Means of an Endpoint Competition Assay. Angew. Chem., Int. Ed. 2015, 54 (47), 1409914102,  DOI: 10.1002/anie.201505800
    22. 22
      Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Activity-Based Protein Profiling: From Enzyme Chemistry to Proteomic Chemistry. Annu. Rev. Biochem. 2008, 77 (1), 383414,  DOI: 10.1146/annurev.biochem.75.101304.124125
    23. 23
      Lanning, B. R.; Whitby, L. R.; Dix, M. M.; Douhan, J.; Gilbert, A. M.; Hett, E. C.; Johnson, T. O.; Joslyn, C.; Kath, J. C.; Niessen, S.; Roberts, L. R.; Schnute, M. E.; Wang, C.; Hulce, J. J.; Wei, B.; Whiteley, L. O.; Hayward, M. M.; Cravatt, B. F. A Road Map to Evaluate the Proteome-Wide Selectivity of Covalent Kinase Inhibitors. Nat. Chem. Biol. 2014, 10 (9), 760767,  DOI: 10.1038/nchembio.1582
    24. 24
      Zaro, B. W.; Whitby, L. R.; Lum, K. M.; Cravatt, B. F. Metabolically Labile Fumarate Esters Impart Kinetic Selectivity to Irreversible Inhibitors. J. Am. Chem. Soc. 2016, 138 (49), 1584115844,  DOI: 10.1021/jacs.6b10589
    25. 25
      Serafimova, I. M.; Pufall, M. A.; Krishnan, S.; Duda, K.; Cohen, M. S.; Maglathlin, R. L.; McFarland, J. M.; Miller, R. M.; Frödin, M.; Taunton, J. Reversible Targeting of Noncatalytic Cysteines with Chemically Tuned Electrophiles. Nat. Chem. Biol. 2012, 8 (5), 471476,  DOI: 10.1038/nchembio.925
    26. 26
      Noe, M. C.; Gilbert, A. M. Targeted Covalent Enzyme Inhibitors. In Annual Reports in Medicinal Chemistry; Desai, M. C., Ed.; Academic Press, 2012; Vol. 47, pp. 413439.  DOI: 10.1016/B978-0-12-396492-2.00027-8 .
    27. 27
      Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.; Jones, L. H.; Gray, N. S. Developing Irreversible Inhibitors of the Protein Kinase Cysteinome. Chem. Biol. 2013, 20 (2), 146159,  DOI: 10.1016/j.chembiol.2012.12.006
    28. 28
      Miller, R. M.; Taunton, J. Targeting Protein Kinases with Selective and Semipromiscuous Covalent Inhibitors. In Methods in Enzymology; Shokat, K. M., Ed.; Academic Press, 2014; Vol. 548, pp 93116.  DOI: 10.1016/B978-0-12-397918-6.00004-5 .
    29. 29
      Gilbert, A. M. Recent Advances in Irreversible Kinase Inhibitors. Pharm. Pat. Anal. 2014, 3 (4), 375386,  DOI: 10.4155/ppa.14.24
    30. 30
      Adeniyi, A. A.; Muthusamy, R.; Soliman, M. E. New Drug Design with Covalent Modifiers. Expert Opin. Drug Discovery 2016, 11 (1), 7990,  DOI: 10.1517/17460441.2016.1115478
    31. 31
      Baillie, T. A. Targeted Covalent Inhibitors for Drug Design. Angew. Chem., Int. Ed. 2016, 55 (43), 1340813421,  DOI: 10.1002/anie.201601091
    32. 32
      Hallenbeck, K. K.; Turner, D. M.; Renslo, A. R.; Arkin, M. R. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery. Curr. Top. Med. Chem. 2016, 17, 415,  DOI: 10.2174/1568026616666160719163839
    33. 33
      Lagoutte, R.; Patouret, R.; Winssinger, N. Covalent Inhibitors: An Opportunity for Rational Target Selectivity. Curr. Opin. Chem. Biol. 2017, 39, 5463,  DOI: 10.1016/j.cbpa.2017.05.008
    34. 34
      De Cesco, S.; Kurian, J.; Dufresne, C.; Mittermaier, A. K.; Moitessier, N. Covalent Inhibitors Design and Discovery. Eur. J. Med. Chem. 2017, 138, 96114,  DOI: 10.1016/j.ejmech.2017.06.019
    35. 35
      Chaikuad, A.; Koch, P.; Laufer, S. A.; Knapp, S. The Cysteinome of Protein Kinases as a Target in Drug Development. Angew. Chem., Int. Ed. 2018, 57 (16), 43724385,  DOI: 10.1002/anie.201707875
    36. 36
      Zhao, Z.; Bourne, P. E. Progress with Covalent Small-Molecule Kinase Inhibitors. Drug Discovery Today 2018, 23 (3), 727735,  DOI: 10.1016/j.drudis.2018.01.035
    37. 37
      Lonsdale, R.; Ward, R. A. Structure-Based Design of Targeted Covalent Inhibitors. Chem. Soc. Rev. 2018, 47 (11), 38163830,  DOI: 10.1039/C7CS00220C
    38. 38
      Ferguson, F. M.; Gray, N. S. Kinase Inhibitors: The Road Ahead. Nat. Rev. Drug Discovery 2018, 17 (5), 353377,  DOI: 10.1038/nrd.2018.21
    39. 39
      Pettinger, J.; Jones, K.; Cheeseman, M. D. Lysine-Targeting Covalent Inhibitors. Angew. Chem., Int. Ed. 2017, 56 (48), 1520015209,  DOI: 10.1002/anie.201707630
    40. 40
      Jackson, P. A.; Widen, J. C.; Harki, D. A.; Brummond, K. M. Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions. J. Med. Chem. 2017, 60 (3), 839885,  DOI: 10.1021/acs.jmedchem.6b00788
    41. 41
      Baslé, E.; Joubert, N.; Pucheault, M. Protein Chemical Modification on Endogenous Amino Acids. Chem. Biol. 2010, 17 (3), 213227,  DOI: 10.1016/j.chembiol.2010.02.008
    42. 42
      Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115 (5), 21742195,  DOI: 10.1021/cr500399p
    43. 43
      Shannon, D. A.; Weerapana, E. Covalent Protein Modification: The Current Landscape of Residue-Specific Electrophiles. Curr. Opin. Chem. Biol. 2015, 24, 1826,  DOI: 10.1016/j.cbpa.2014.10.021
    44. 44
      Gunnoo, S. B.; Madder, A. Chemical Protein Modification through Cysteine. ChemBioChem 2016, 17 (7), 529553,  DOI: 10.1002/cbic.201500667
    45. 45
      Dondoni, A.; Marra, A. SuFEx: A Metal-Free Click Ligation for Multivalent Biomolecules. Org. Biomol. Chem. 2017, 15 (7), 15491553,  DOI: 10.1039/C6OB02458K
    46. 46
      deGruyter, J. N.; Malins, L. R.; Baran, P. S. Residue-Specific Peptide Modification: A Chemist’s Guide. Biochemistry 2017, 56 (30), 38633873,  DOI: 10.1021/acs.biochem.7b00536
    47. 47
      Hoch, D. G.; Abegg, D.; Adibekian, A. Cysteine-Reactive Probes and Their Use in Chemical Proteomics. Chem. Commun. 2018, 54 (36), 45014512,  DOI: 10.1039/C8CC01485J
    48. 48
      Cromm, P. M.; Crews, C. M. The Proteasome in Modern Drug Discovery: Second Life of a Highly Valuable Drug Target. ACS Cent. Sci. 2017, 3 (8), 830838,  DOI: 10.1021/acscentsci.7b00252
    49. 49
      Casimiro-Garcia, A.; Trujillo, J. I.; Vajdos, F.; Juba, B.; Banker, M. E.; Aulabaugh, A.; Balbo, P.; Bauman, J.; Chrencik, J.; Coe, J. W.; Czerwinski, R.; Dowty, M.; Knafels, J. D.; Kwon, S.; Leung, L.; Liang, S.; Robinson, R. P.; Telliez, J.-B.; Unwalla, R.; Yang, X.; Thorarensen, A. Identification of Cyanamide-Based Janus Kinase 3 (JAK3) Covalent Inhibitors. J. Med. Chem. 2018, 61, 1066510699,  DOI: 10.1021/acs.jmedchem.8b01308
    50. 50
      Li, T.; Maltais, R.; Poirier, D.; Lin, S.-X. Combined Biophysical Chemistry Reveals a New Covalent Inhibitor with a Low-Reactivity Alkyl Halide. J. Phys. Chem. Lett. 2018, 9 (18), 52755280,  DOI: 10.1021/acs.jpclett.8b02225
    51. 51
      Kharenko, O. A.; Patel, R. G.; Brown, S. D.; Calosing, C.; White, A.; Lakshminarasimhan, D.; Suto, R. K.; Duffy, B. C.; Kitchen, D. B.; McLure, K. G.; Hansen, H. C.; van der Horst, E. H.; Young, P. R. Design and Characterization of Novel Covalent Bromodomain and Extra-Terminal Domain (BET) Inhibitors Targeting a Methionine. J. Med. Chem. 2018, 61 (18), 82028211,  DOI: 10.1021/acs.jmedchem.8b00666
    52. 52
      Pearson, R. G.; Sobel, H. R.; Songstad, J. Nucleophilic Reactivity Constants toward Methyl Iodide and Trans-Dichlorodi(Pyridine)Platinum(II). J. Am. Chem. Soc. 1968, 90 (2), 319326,  DOI: 10.1021/ja01004a021
    53. 53
      Sardi, F.; Manta, B.; Portillo-Ledesma, S.; Knoops, B.; Comini, M. A.; Ferrer-Sueta, G. Determination of Acidity and Nucleophilicity in Thiols by Reaction with Monobromobimane and Fluorescence Detection. Anal. Biochem. 2013, 435 (1), 7482,  DOI: 10.1016/j.ab.2012.12.017
    54. 54
      Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc. 1963, 85 (22), 35333539,  DOI: 10.1021/ja00905a001
    55. 55
      Awoonor-Williams, E.; Rowley, C. N. Evaluation of Methods for the Calculation of the PKa of Cysteine Residues in Proteins. J. Chem. Theory Comput. 2016, 12 (9), 46624673,  DOI: 10.1021/acs.jctc.6b00631
    56. 56
      Grimsley, G. R.; Scholtz, J. M.; Pace, C. N. A Summary of the Measured PK Values of the Ionizable Groups in Folded Proteins. Protein Sci. 2008, 18, 247251,  DOI: 10.1002/pro.19
    57. 57
      Alcock, L. J.; Perkins, M. V.; Chalker, J. M. Chemical Methods for Mapping Cysteine Oxidation. Chem. Soc. Rev. 2018, 47 (1), 231268,  DOI: 10.1039/C7CS00607A
    58. 58
      Pace, N. J.; Weerapana, E. Diverse Functional Roles of Reactive Cysteines. ACS Chem. Biol. 2013, 8 (2), 283296,  DOI: 10.1021/cb3005269
    59. 59
      Jones, A.; Zhang, X.; Lei, X. Covalent Probe Finds Carboxylic Acid. Cell Chem. Biol. 2017, 24 (5), 537539,  DOI: 10.1016/j.chembiol.2017.05.003
    60. 60
      Shirley, M. Dacomitinib: First Global Approval. Drugs 2018, 78, 1947,  DOI: 10.1007/s40265-018-1028-x
    61. 61
      Markham, A.; Dhillon, S. Acalabrutinib: First Global Approval. Drugs 2018, 78 (1), 139145,  DOI: 10.1007/s40265-017-0852-8
    62. 62
      Forster, M.; Gehringer, M.; Laufer, S. A. Recent Advances in JAK3 Inhibition: Isoform Selectivity by Covalent Cysteine Targeting. Bioorg. Med. Chem. Lett. 2017, 27 (18), 42294237,  DOI: 10.1016/j.bmcl.2017.07.079
    63. 63
      Garzón, B.; Oeste, C. L.; Díez-Dacal, B.; Pérez-Sala, D. Proteomic Studies on Protein Modification by Cyclopentenone Prostaglandins: Expanding Our View on Electrophile Actions. J. Proteomics 2011, 74 (11), 22432263,  DOI: 10.1016/j.jprot.2011.03.028
    64. 64
      Zhao, Z.; Liu, Q.; Bliven, S.; Xie, L.; Bourne, P. E. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome. J. Med. Chem. 2017, 60 (7), 28792889,  DOI: 10.1021/acs.jmedchem.6b01815
    65. 65
      Günther, M.; Juchum, M.; Kelter, G.; Fiebig, H.; Laufer, S. Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant. Angew. Chem., Int. Ed. 2016, 55 (36), 1089010894,  DOI: 10.1002/anie.201603736
    66. 66
      Niessen, S.; Dix, M. M.; Barbas, S.; Potter, Z. E.; Lu, S.; Brodsky, O.; Planken, S.; Behenna, D.; Almaden, C.; Gajiwala, K. S.; Ryan, K.; Ferre, R.; Lazear, M. R.; Hayward, M. M.; Kath, J. C.; Cravatt, B. F. Proteome-Wide Map of Targets of T790M-EGFR-Directed Covalent Inhibitors. Cell Chem. Biol. 2017, 24, 13881400,  DOI: 10.1016/j.chembiol.2017.08.017
    67. 67
      Blewett, M. M.; Xie, J.; Zaro, B. W.; Backus, K. M.; Altman, A.; Teijaro, J. R.; Cravatt, B. F. Chemical Proteomic Map of Dimethyl Fumarate–Sensitive Cysteines in Primary Human T Cells. Sci. Signaling 2016, 9 (445), rs10,  DOI: 10.1126/scisignal.aaf7694
    68. 68
      Deeks, E. D. Ibrutinib: A Review in Chronic Lymphocytic Leukaemia. Drugs 2017, 77 (2), 225236,  DOI: 10.1007/s40265-017-0695-3
    69. 69
      Bender, A. T.; Gardberg, A.; Pereira, A.; Johnson, T.; Wu, Y.; Grenningloh, R.; Head, J.; Morandi, F.; Haselmayer, P.; Liu-Bujalski, L. Ability of Bruton’s Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but Not B-Cell Receptor Signaling. Mol. Pharmacol. 2017, 91 (3), 208219,  DOI: 10.1124/mol.116.107037
    70. 70
      Pan, Z.; Scheerens, H.; Li, S.-J.; Schultz, B. E.; Sprengeler, P. A.; Burrill, L. C.; Mendonca, R. V.; Sweeney, M. D.; Scott, K. C. K.; Grothaus, P. G.; Jeffery, D. A.; Spoerke, J. M.; Honigberg, L. A.; Young, P. R.; Dalrymple, S. A.; Palmer, J. T. Discovery of Selective Irreversible Inhibitors for Bruton’s Tyrosine Kinase. ChemMedChem 2007, 2 (1), 5861,  DOI: 10.1002/cmdc.200600221
    71. 71
      Mann, M. Innovations: Functional and Quantitative Proteomics Using SILAC. Nat. Rev. Mol. Cell Biol. 2006, 7 (12), 952958,  DOI: 10.1038/nrm2067
    72. 72
      Crow, J. A.; Bittles, V.; Borazjani, A.; Potter, P. M.; Ross, M. K. Covalent Inhibition of Recombinant Human Carboxylesterase 1 and 2 and Monoacylglycerol Lipase by the Carbamates JZL184 and URB597. Biochem. Pharmacol. 2012, 84 (9), 12151222,  DOI: 10.1016/j.bcp.2012.08.017
    73. 73
      Buynak, J. D.; Mathew, J.; Rao, M. N.; Haley, E.; George, C.; Siriwardane, U. The Preparation of the First α-Vinylidene-β-Lactams. J. Chem. Soc., Chem. Commun. 1987, 0 (10), 735737,  DOI: 10.1039/C39870000735
    74. 74
      Roedig, A.; Ritschel, W. Reaktionen von 3,4,4-Trichlor-3-butenamiden mit Nucleophilen, II. Thiol- und Aminaddukte von 3,3-Dichlorallencarboxamiden. Chem. Ber. 1983, 116 (4), 15951602,  DOI: 10.1002/cber.19831160434
    75. 75
      Abbas, A.; Xing, B.; Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem., Int. Ed. 2014, 53 (29), 74917494,  DOI: 10.1002/anie.201403121
    76. 76
      Pedzisa, L.; Li, X.; Rader, C.; Roush, W. R. Assessment of Reagents for Selenocysteine Conjugation and the Stability of Selenocysteine Adducts. Org. Biomol. Chem. 2016, 14 (22), 51415147,  DOI: 10.1039/C6OB00775A
    77. 77
      Chen, D.; Guo, D.; Yan, Z.; Zhao, Y. Allenamide as a Bioisostere of Acrylamide in the Design and Synthesis of Targeted Covalent Inhibitors. MedChemComm 2018, 9 (2), 244253,  DOI: 10.1039/C7MD00571G
    78. 78
      Awoonor-Williams, E.; Rowley, C. N. How Reactive Are Druggable Cysteines in Protein Kinases?. J. Chem. Inf. Model. 2018, 58 (9), 19351946,  DOI: 10.1021/acs.jcim.8b00454
    79. 79
      Koniev, O.; Leriche, G.; Nothisen, M.; Remy, J.-S.; Strub, J.-M.; Schaeffer-Reiss, C.; Van Dorsselaer, A.; Baati, R.; Wagner, A. Selective Irreversible Chemical Tagging of Cysteine with 3-Arylpropiolonitriles. Bioconjugate Chem. 2014, 25 (2), 202206,  DOI: 10.1021/bc400469d
    80. 80
      Shiu, H.-Y.; Chan, T.-C.; Ho, C.-M.; Liu, Y.; Wong, M.-K.; Che, C.-M. Electron-Deficient Alkynes as Cleavable Reagents for the Modification of Cysteine-Containing Peptides in Aqueous Medium. Chem. - Eur. J. 2009, 15 (15), 38393850,  DOI: 10.1002/chem.200800669
    81. 81
      Friedman, M.; Wall, J. S. Additive Linear Free-Energy Relationships in Reaction Kinetics of Amino Groups with α,β-Unsaturated Compounds. J. Org. Chem. 1966, 31 (9), 28882894,  DOI: 10.1021/jo01347a036
    82. 82
      Cavins, J. F.; Friedman, M. An Internal Standard for Amino Acid Analyses: S-β-(4-Pyridylethyl)-l-Cysteine. Anal. Biochem. 1970, 35 (2), 489493,  DOI: 10.1016/0003-2697(70)90211-3
    83. 83
      Gill, A. L.; Frederickson, M.; Cleasby, A.; Woodhead, S. J.; Carr, M. G.; Woodhead, A. J.; Walker, M. T.; Congreve, M. S.; Devine, L. A.; Tisi, D.; O’Reilly, M.; Seavers, L. C. A.; Davis, D. J.; Curry, J.; Anthony, R.; Padova, A.; Murray, C. W.; Carr, R. A. E.; Jhoti, H. Identification of Novel P38α MAP Kinase Inhibitors Using Fragment-Based Lead Generation. J. Med. Chem. 2005, 48 (2), 414426,  DOI: 10.1021/jm049575n
    84. 84
      Raux, E.; Klenc, J.; Blake, A.; Sączewski, J.; Strekowski, L. Conjugate Addition of Nucleophiles to the Vinyl Function of 2-Chloro-4-Vinylpyrimidine Derivatives. Molecules 2010, 15 (3), 19731984,  DOI: 10.3390/molecules15031973
    85. 85
      Burns, A. R.; Kerr, J. H.; Kerr, W. J.; Passmore, J.; Paterson, L. C.; Watson, A. J. B. Tuned Methods for Conjugate Addition to a Vinyl Oxadiazole; Synthesis of Pharmaceutically Important Motifs. Org. Biomol. Chem. 2010, 8 (12), 27772783,  DOI: 10.1039/c001772h
    86. 86
      Kuchař, M.; Hocek, M.; Pohl, R.; Votruba, I.; Shih, I.; Mabery, E.; Mackman, R. Synthesis, Cytostatic, and Antiviral Activity of Novel 6-[2-(Dialkylamino)Ethyl]-, 6-(2-Alkoxyethyl)-, 6-[2-(Alkylsulfanyl)Ethyl]-, and 6-[2-(Dialkylamino)Vinyl]Purine Nucleosides. Bioorg. Med. Chem. 2008, 16 (3), 14001424,  DOI: 10.1016/j.bmc.2007.10.063
    87. 87
      Il’yasov, E. A.; Galust’yan, G. G. Homolytic Addition of 1-Alkanethiols to 5-Ethynyl-2-Methylpyridine. Chem. Heterocycl. Compd. 1999, 35 (10), 11871189,  DOI: 10.1007/BF02323377
    88. 88
      Wipf, P.; Graham, T. H. Synthesis and Hetero-Michael Addition Reactions of 2-Alkynyl Oxazoles and Oxazolines. Org. Biomol. Chem. 2005, 3 (1), 3135,  DOI: 10.1039/b413604g
    89. 89
      Li, Q.-F.; Yang, Y.; Maleckis, A.; Otting, G.; Su, X.-C. Thiol–Ene Reaction: A Versatile Tool in Site-Specific Labelling of Proteins with Chemically Inert Tags for Paramagnetic NMR. Chem. Commun. 2012, 48 (21), 27042706,  DOI: 10.1039/c2cc17900h
    90. 90
      Yang, Y.; Li, Q.-F.; Cao, C.; Huang, F.; Su, X.-C. Site-Specific Labeling of Proteins with a Chemically Stable, High-Affinity Tag for Protein Study. Chem. - Eur. J. 2013, 19 (3), 10971103,  DOI: 10.1002/chem.201202495
    91. 91
      Ma, F.-H.; Chen, J.-L.; Li, Q.-F.; Zuo, H.-H.; Huang, F.; Su, X.-C. Kinetic Assay of the Michael Addition-Like Thiol–Ene Reaction and Insight into Protein Bioconjugation. Chem. - Asian J. 2014, 9 (7), 18081816,  DOI: 10.1002/asia.201402095
    92. 92
      Wood, E. R.; Shewchuk, L. M.; Ellis, B.; Brignola, P.; Brashear, R. L.; Caferro, T. R.; Dickerson, S. H.; Dickson, H. D.; Donaldson, K. H.; Gaul, M.; Griffin, R. J.; Hassell, A. M.; Keith, B.; Mullin, R.; Petrov, K. G.; Reno, M. J.; Rusnak, D. W.; Tadepalli, S. M.; Ulrich, J. C.; Wagner, C. D.; Vanderwall, D. E.; Waterson, A. G.; Williams, J. D.; White, W. L.; Uehling, D. E. 6-Ethynylthieno[3,2-d]- and 6-Ethynylthieno[2,3-d]Pyrimidin-4-Anilines as Tunable Covalent Modifiers of ErbB Kinases. Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (8), 27732778,  DOI: 10.1073/pnas.0708281105
    93. 93
      Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A. Tyrosine Kinase Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth Factor Receptor:  4-(Phenylamino)Quinazoline- and 4-(Phenylamino)Pyrido[3,2-d]Pyrimidine-6-Acrylamides Bearing Additional Solubilizing Functions. J. Med. Chem. 2000, 43 (7), 13801397,  DOI: 10.1021/jm990482t
    94. 94
      Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Greenberger, L. M.; Wang, Y.-F.; Wissner, A. 6-Substituted-4-(3-Bromophenylamino)Quinazolines as Putative Irreversible Inhibitors of the Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor Activity. J. Med. Chem. 2001, 44 (17), 27192734,  DOI: 10.1021/jm0005555
    95. 95
      Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi, X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.; Tsou, H.-R. Synthesis and Structure–Activity Relationships of 6,7-Disubstituted 4-Anilinoquinoline-3-Carbonitriles. The Design of an Orally Active, Irreversible Inhibitor of the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor (EGFR) and the Human Epidermal Growth Factor Receptor-2 (HER-2). J. Med. Chem. 2003, 46 (1), 4963,  DOI: 10.1021/jm020241c
    96. 96
      Hubbard, R. D.; Dickerson, S. H.; Emerson, H. K.; Griffin, R. J.; Reno, M. J.; Hornberger, K. R.; Rusnak, D. W.; Wood, E. R.; Uehling, D. E.; Waterson, A. G. Dual EGFR/ErbB-2 Inhibitors from Novel Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines. Bioorg. Med. Chem. Lett. 2008, 18 (21), 57385740,  DOI: 10.1016/j.bmcl.2008.09.090
    97. 97
      Stevens, K. L.; Alligood, K. J.; Alberti, J. G. B.; Caferro, T. R.; Chamberlain, S. D.; Dickerson, S. H.; Dickson, H. D.; Emerson, H. K.; Griffin, R. J.; Hubbard, R. D.; Keith, B. R.; Mullin, R. J.; Petrov, K. G.; Gerding, R. M.; Reno, M. J.; Rheault, T. R.; Rusnak, D. W.; Sammond, D. M.; Smith, S. C.; Uehling, D. E.; Waterson, A. G.; Wood, E. R. Synthesis and Stereochemical Effects of Pyrrolidinyl-Acetylenic Thieno[3,2-d]Pyrimidines as EGFR and ErbB-2 Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (1), 2126,  DOI: 10.1016/j.bmcl.2008.11.023
    98. 98
      Waterson, A. G.; Petrov, K. G.; Hornberger, K. R.; Hubbard, R. D.; Sammond, D. M.; Smith, S. C.; Dickson, H. D.; Caferro, T. R.; Hinkle, K. W.; Stevens, K. L.; Dickerson, S. H.; Rusnak, D. W.; Spehar, G. M.; Wood, E. R.; Griffin, R. J.; Uehling, D. E. Synthesis and Evaluation of Aniline Headgroups for Alkynyl Thienopyrimidine Dual EGFR/ErbB-2 Kinase Inhibitors. Bioorg. Med. Chem. Lett. 2009, 19 (5), 13321336,  DOI: 10.1016/j.bmcl.2009.01.080
    99. 99
      Nijmeijer, S.; Engelhardt, H.; Schultes, S.; van de Stolpe, A. C.; Lusink, V.; de Graaf, C.; Wijtmans, M.; Haaksma, E. E. J.; de Esch, I. J. P.; Stachurski, K.; Vischer, H. F.; Leurs, R. Design and Pharmacological Characterization of VUF14480, a Covalent Partial Agonist That Interacts with Cysteine 983.36 of the Human Histamine H4 Receptor. Br. J. Pharmacol. 2013, 170, 89100,  DOI: 10.1111/bph.12113
    100. 100
      Schapira, A.; Bate, G.; Kirkpatrick, P. Rasagiline. Nat. Rev. Drug Discovery 2005, 4 (8), 625626,  DOI: 10.1038/nrd1803
    101. 101
      Youdim, M. B. H.; Gross, A.; Finberg, J. P. M. Rasagiline [N-Propargyl-1R(+)-Aminoindan], a Selective and Potent Inhibitor of Mitochondrial Monoamine Oxidase B. Br. J. Pharmacol. 2001, 132 (2), 500506,  DOI: 10.1038/sj.bjp.0703826
    102. 102
      Wright, A. T.; Song, J. D.; Cravatt, B. F. A Suite of Activity-Based Probes for Human Cytochrome P450 Enzymes. J. Am. Chem. Soc. 2009, 131 (30), 1069210700,  DOI: 10.1021/ja9037609
    103. 103
      Wright, A. T.; Cravatt, B. F. Chemical Proteomic Probes for Profiling Cytochrome P450 Activities and Drug Interactions In Vivo. Chem. Biol. 2007, 14 (9), 10431051,  DOI: 10.1016/j.chembiol.2007.08.008
    104. 104
      van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C. Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition. Bioconjugate Chem. 2012, 23 (3), 392398,  DOI: 10.1021/bc200365k
    105. 105
      Tian, H.; Sakmar, T. P.; Huber, T. A Simple Method for Enhancing the Bioorthogonality of Cyclooctyne Reagent. Chem. Commun. 2016, 52 (31), 54515454,  DOI: 10.1039/C6CC01321J
    106. 106
      Haldón, E.; Nicasio, M. C.; Pérez, P. J. Copper-Catalysed Azide–Alkyne Cycloadditions (CuAAC): An Update. Org. Biomol. Chem. 2015, 13 (37), 95289550,  DOI: 10.1039/C5OB01457C
    107. 107
      Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.; Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.; Heck, A. J. R.; Komander, D.; Ovaa, H. On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases. J. Am. Chem. Soc. 2013, 135 (8), 28672870,  DOI: 10.1021/ja309802n
    108. 108
      Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D. Covalent Inhibition of SUMO and Ubiquitin-Specific Cysteine Proteases by an in Situ Thiol–Alkyne Addition. Bioorg. Med. Chem. 2013, 21 (9), 25112517,  DOI: 10.1016/j.bmc.2013.02.039
    109. 109
      Swatek, K. N.; Aumayr, M.; Pruneda, J. N.; Visser, L. J.; Berryman, S.; Kueck, A. F.; Geurink, P. P.; Ovaa, H.; van Kuppeveld, F. J. M.; Tuthill, T. J.; Skern, T.; Komander, D. Irreversible Inactivation of ISG15 by a Viral Leader Protease Enables Alternative Infection Detection Strategies. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (10), 23712376,  DOI: 10.1073/pnas.1710617115
    110. 110
      Arkona, C.; Rademann, J. Propargyl Amides as Irreversible Inhibitors of Cysteine Proteases—A Lesson on the Biological Reactivity of Alkynes. Angew. Chem., Int. Ed. 2013, 52 (32), 82108212,  DOI: 10.1002/anie.201303544
    111. 111
      Sanger, F. The Free Amino Groups of Insulin. Biochem. J. 1945, 39 (5), 507515,  DOI: 10.1042/bj0390507
    112. 112
      Terrier, F. Rate and Equilibrium Studies in Jackson-Meisenheimer Complexes. Chem. Rev. 1982, 82 (2), 77152,  DOI: 10.1021/cr00048a001
    113. 113
      Terrier, F. Modern Nucleophilic Aromatic Substitution, 1st ed.; Wiley-VCH: Weinheim, 2013.
    114. 114
      Kwan, E. E.; Zeng, Y.; Besser, H. A.; Jacobsen, E. N. Concerted Nucleophilic Aromatic Substitutions. Nat. Chem. 2018, 10 (9), 917923,  DOI: 10.1038/s41557-018-0079-7
    115. 115
      Elbrecht, A.; Chen, Y.; Adams, A.; Berger, J.; Griffin, P.; Klatt, T.; Zhang, B.; Menke, J.; Zhou, G.; Smith, R. G.; Moller, D. E. L-764406 Is a Partial Agonist of Human Peroxisome Proliferator-Activated Receptor γ. The Role of Cys13 in Ligand Binding. J. Biol. Chem. 1999, 274 (12), 79137922,  DOI: 10.1074/jbc.274.12.7913
    116. 116
      Leesnitzer, L. M.; Parks, D. J.; Bledsoe, R. K.; Cobb, J. E.; Collins, J. L.; Consler, T. G.; Davis, R. G.; Hull-Ryde, E. A.; Lenhard, J. M.; Patel, L.; Plunket, K. D.; Shenk, J. L.; Stimmel, J. B.; Therapontos, C.; Willson, T. M.; Blanchard, S. G. Functional Consequences of Cysteine Modification in the Ligand Binding Sites of Peroxisome Proliferator Activated Receptors by GW9662. Biochemistry 2002, 41 (21), 66406650,  DOI: 10.1021/bi0159581
    117. 117
      Shearer, B. G.; Wiethe, R. W.; Ashe, A.; Billin, A. N.; Way, J. M.; Stanley, T. B.; Wagner, C. D.; Xu, R. X.; Leesnitzer, L. M.; Merrihew, R. V.; Shearer, T. W.; Jeune, M. R.; Ulrich, J. C.; Willson, T. M. Identification and Characterization of 4-Chloro-N-(2-{[5-Trifluoromethyl)-2-Pyridyl]Sulfonyl}ethyl)Benzamide (GSK3787), a Selective and Irreversible Peroxisome Proliferator-Activated Receptor δ (PPARδ) Antagonist. J. Med. Chem. 2010, 53 (4), 18571861,  DOI: 10.1021/jm900464j
    118. 118
      Babaoglu, K.; Simeonov, A.; Irwin, J. J.; Nelson, M. E.; Feng, B.; Thomas, C. J.; Cancian, L.; Costi, M. P.; Maltby, D. A.; Jadhav, A.; Inglese, J.; Austin, C. P.; Shoichet, B. K. Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase. J. Med. Chem. 2008, 51 (8), 25022511,  DOI: 10.1021/jm701500e
    119. 119
      Patterson, J. T.; Asano, S.; Li, X.; Rader, C.; Barbas, C. F. Improving the Serum Stability of Site-Specific Antibody Conjugates with Sulfone Linkers. Bioconjugate Chem. 2014, 25 (8), 14021407,  DOI: 10.1021/bc500276m
    120. 120
      Zhang, D.; Devarie-Baez, N. O.; Li, Q.; Lancaster, J. R.; Xian, M. Methylsulfonyl Benzothiazole (MSBT): A Selective Protein Thiol Blocking Reagent. Org. Lett. 2012, 14 (13), 33963399,  DOI: 10.1021/ol301370s
    121. 121
      Toda, N.; Asano, S.; Barbas, C. F. Rapid, Stable, Chemoselective Labeling of Thiols with Julia–Kocieński-like Reagents: A Serum-Stable Alternative to Maleimide-Based Protein Conjugation. Angew. Chem., Int. Ed. 2013, 52 (48), 1259212596,  DOI: 10.1002/anie.201306241
    122. 122
      Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y.-S.; Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135 (16), 59465949,  DOI: 10.1021/ja400119t
    123. 123
      Alapour, S.; de la Torre, B. G.; Ramjugernath, D.; Koorbanally, N. A.; Albericio, F. Application of Decafluorobiphenyl (DFBP) Moiety as a Linker in Bioconjugation. Bioconjugate Chem. 2018, 29 (2), 225233,  DOI: 10.1021/acs.bioconjchem.7b00800
    124. 124
      Brown, S. P.; Smith, A. B. Peptide/Protein Stapling and Unstapling: Introduction of s-Tetrazine, Photochemical Release, and Regeneration of the Peptide/Protein. J. Am. Chem. Soc. 2015, 137 (12), 40344037,  DOI: 10.1021/ja512880g
    125. 125
      Roberts, D. W.; Aptula, A. O. Electrophilic Reactivity and Skin Sensitization Potency of SNAr Electrophiles. Chem. Res. Toxicol. 2014, 27 (2), 240246,  DOI: 10.1021/tx400355n
    126. 126
      Hwang, J. Y.; Huang, W.; Arnold, L. A.; Huang, R.; Attia, R. R.; Connelly, M.; Wichterman, J.; Zhu, F.; Augustinaite, I.; Austin, C. P.; Inglese, J.; Johnson, R. L.; Guy, R. K. Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone. J. Biol. Chem. 2011, 286 (14), 1189511908,  DOI: 10.1074/jbc.M110.200436
    127. 127
      Arnold, L. A.; Kosinski, A.; Estébanez-Perpiñá, E.; Guy, R. K. Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators:  Preliminary Structure–Activity Relationships. J. Med. Chem. 2007, 50 (22), 52695280,  DOI: 10.1021/jm070556y
    128. 128
      Visperas, P. R.; Winger, J. A.; Horton, T. M.; Shah, N. H.; Aum, D. J.; Tao, A.; Barros, T.; Yan, Q.; Wilson, C. G.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Modification by Covalent Reaction or Oxidation of Cysteine Residues in the Tandem-SH2 Domains of ZAP-70 and Syk Can Block Phosphopeptide Binding. Biochem. J. 2015, 465 (1), 149161,  DOI: 10.1042/BJ20140793
    129. 129
      Visperas, P. R.; Wilson, C. G.; Winger, J. A.; Yan, Q.; Lin, K.; Arkin, M. R.; Weiss, A.; Kuriyan, J. Identification of Inhibitors of the Association of ZAP-70 with the T Cell Receptor by High-Throughput Screen. SLAS Discov. 2017, 22 (3), 324331,  DOI: 10.1177/1087057116681407
    130. 130
      Shannon, D. A.; Banerjee, R.; Webster, E. R.; Bak, D. W.; Wang, C.; Weerapana, E. Investigating the Proteome Reactivity and Selectivity of Aryl Halides. J. Am. Chem. Soc. 2014, 136 (9), 33303333,  DOI: 10.1021/ja4116204
    131. 131
      Schardon, C. L.; Tuley, A.; Er, J. A. V.; Swartzel, J. C.; Fast, W. Selective Covalent Protein Modification by 4-Halopyridines through Catalysis. ChemBioChem 2017, 18 (15), 15511556,  DOI: 10.1002/cbic.201700104
    132. 132
      Fairhurst, R. A.; Knoepfel, T.; Leblanc, C.; Buschmann, N.; Gaul, C.; Blank, J.; Galuba, I.; Trappe, J.; Zou, C.; Voshol, J.; Genick, C.; Brunet-Lefeuvre, P.; Bitsch, F.; Graus-Porta, D.; Furet, P. Approaches to Selective Fibroblast Growth Factor Receptor 4 Inhibition through Targeting the ATP-Pocket Middle-Hinge Region. MedChemComm 2017, 8, 16041613,  DOI: 10.1039/C7MD00213K
    133. 133
      Hou, W.; Ren, Y.; Zhang, Z.; Sun, H.; Ma, Y.; Yan, B. Novel Quinazoline Derivatives Bearing Various 6-Benzamide Moieties as Highly Selective and Potent EGFR Inhibitors. Bioorg. Med. Chem. 2018, 26 (8), 17401750,  DOI: 10.1016/j.bmc.2018.02.022
    134. 134
      Juchum, M.; Günther, M.; Laufer, S. A. Fighting Cancer Drug Resistance: Opportunities and Challenges for Mutation-Specific EGFR Inhibitors. Drug Resist. Updates 2015, 20, 1228,  DOI: 10.1016/j.drup.2015.05.002
    135. 135
      Chen, K. X.; Lesburg, C. A.; Vibulbhan, B.; Yang, W.; Chan, T.-Y.; Venkatraman, S.; Velazquez, F.; Zeng, Q.; Bennett, F.; Anilkumar, G. N.; Duca, J.; Jiang, Y.; Pinto, P.; Wang, L.; Huang, Y.; Selyutin, O.; Gavalas, S.; Pu, H.; Agrawal, S.; Feld, B.; Huang, H.-C.; Li, C.; Cheng, K.-C.; Shih, N.-Y.; Kozlowski, J. A.; Rosenblum, S. B.; Njoroge, F. G. A Novel Class of Highly Potent Irreversible Hepatitis C Virus NS5B Polymerase Inhibitors. J. Med. Chem. 2012, 55 (5), 20892101,  DOI: 10.1021/jm201322r
    136. 136
      Powers, J. P.; Piper, D. E.; Li, Y.; Mayorga, V.; Anzola, J.; Chen, J. M.; Jaen, J. C.; Lee, G.; Liu, J.; Peterson, M. G.; Tonn, G. R.; Ye, Q.; Walker, N. P. C.; Wang, Z. SAR and Mode of Action of Novel Non-Nucleoside Inhibitors of Hepatitis C NS5b RNA Polymerase. J. Med. Chem. 2006, 49 (3), 10341046,  DOI: 10.1021/jm050859x
    137. 137
      Huth, J. R.; Mendoza, R.; Olejniczak, E. T.; Johnson, R. W.; Cothron, D. A.; Liu, Y.; Lerner, C. G.; Chen, J.; Hajduk, P. J. ALARM NMR:  A Rapid and Robust Experimental Method to Detect Reactive False Positives in Biochemical Screens. J. Am. Chem. Soc. 2005, 127 (1), 217224,  DOI: 10.1021/ja0455547
    138. 138
      Boelsterli, U. A.; Ho, H. K.; Zhou, S.; Leow, K. Y. Bioactivation and Hepatotoxicity of Nitroaromatic Drugs. Curr. Drug Metab. 2006, 7, 715727,  DOI: 10.2174/138920006778520606
    139. 139
      Zeng, Q.; Nair, A. G.; Rosenblum, S. B.; Huang, H.-C.; Lesburg, C. A.; Jiang, Y.; Selyutin, O.; Chan, T.-Y.; Bennett, F.; Chen, K. X.; Venkatraman, S.; Sannigrahi, M.; Velazquez, F.; Duca, J. S.; Gavalas, S.; Huang, Y.; Pu, H.; Wang, L.; Pinto, P.; Vibulbhan, B.; Agrawal, S.; Ferrari, E.; Jiang, C.; Li, C.; Hesk, D.; Gesell, J.; Sorota, S.; Shih, N.-Y.; Njoroge, F. G.; Kozlowski, J. A. Discovery of an Irreversible HCV NS5B Polymerase Inhibitor. Bioorg. Med. Chem. Lett. 2013, 23 (24), 65856587,  DOI: 10.1016/j.bmcl.2013.10.060
    140. 140
      Sato, K.; Kunitomo, Y.; Kasai, Y.; Utsumi, S.; Suetake, I.; Tajima, S.; Ichikawa, S.; Matsuda, A. Mechanism-Based Inhibitor of DNA Cytosine-5 Methyltransferase by a SNAr Reaction with an Oligodeoxyribonucleotide Containing a 2-Amino-4-Halopyridine-C-Nucleoside. ChemBioChem 2018, 19 (8), 865872,  DOI: 10.1002/cbic.201700688
    141. 141
      Kasai, Y.; Sato, K.; Utsumi, S.; Ichikawa, S. Improvement of SNAr Reaction Rate by an Electron-Withdrawing Group in the Crosslinking of DNA Cytosine-5 Methyltransferase by a Covalent Oligodeoxyribonucleotide Inhibitor. ChemBioChem 2018, 19 (17), 18661872,  DOI: 10.1002/cbic.201800244
    142. 142
      Gianatassio, R.; Lopchuk, J. M.; Wang, J.; Pan, C.-M.; Malins, L. R.; Prieto, L.; Brandt, T. A.; Collins, M. R.; Gallego, G. M.; Sach, N. W.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Amination. Science 2016, 351 (6270), 241246,  DOI: 10.1126/science.aad6252
    143. 143
      Lopchuk, J. M.; Fjelbye, K.; Kawamata, Y.; Malins, L. R.; Pan, C.-M.; Gianatassio, R.; Wang, J.; Prieto, L.; Bradow, J.; Brandt, T. A.; Collins, M. R.; Elleraas, J.; Ewanicki, J.; Farrell, W.; Fadeyi, O. O.; Gallego, G. M.; Mousseau, J. J.; Oliver, R.; Sach, N. W.; Smith, J. K.; Spangler, J. E.; Zhu, H.; Zhu, J.; Baran, P. S. Strain-Release Heteroatom Functionalization: Development, Scope, and Stereospecificity. J. Am. Chem. Soc. 2017, 139 (8), 32093226,  DOI: 10.1021/jacs.6b13229
    144. 144
      Semmler, K.; Szeimies, G.; Belzner, J. Tetracyclo[5.1.0.01,6.02,7]Octane, a [1.1.1]Propellane Derivative, and a New Route to the Parent Hydrocarbon. J. Am. Chem. Soc. 1985, 107 (22), 64106411,  DOI: 10.1021/ja00308a053
    145. 145
      Haag, B.; Mosrin, M.; Ila, H.; Malakhov, V.; Knochel, P. Regio- and Chemoselective Metalation of Arenes and Heteroarenes Using Hindered Metal Amide Bases. Angew. Chem., Int. Ed. 2011, 50 (42), 97949824,  DOI: 10.1002/anie.201101960
    146. 146
      Wishart, D. S.; Feunang, Y. D.; Guo, A. C.; Lo, E. J.; Marcu, A.; Grant, J. R.; Sajed, T.; Johnson, D.; Li, C.; Sayeeda, Z.; Assempour, N.; Iynkkaran, I.; Liu, Y.; Maciejewski, A.; Gale, N.; Wilson, A.; Chin, L.; Cummings, R.; Le, D.; Pon, A.; Knox, C.; Wilson, M. DrugBank 5.0: A Major Update to the DrugBank Database for 2018. Nucleic Acids Res. 2018, 46 (D1), D1074D1082,  DOI: 10.1093/nar/gkx1037
    147. 147
      Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of Fluorine in Medicinal Chemistry. J. Med. Chem. 2015, 58 (21), 83158359,  DOI: 10.1021/acs.jmedchem.5b00258
    148. 148
      Meanwell, N. A. Fluorine and Fluorinated Motifs in the Design and Application of Bioisosteres for Drug Design. J. Med. Chem. 2018, 61 (14), 58225880,  DOI: 10.1021/acs.jmedchem.7b01788
    149. 149
      Grishin, N. V.; Osterman, A. L.; Brooks, H. B.; Phillips, M. A.; Goldsmith, E. J. X-Ray Structure of Ornithine Decarboxylase from Trypanosoma Brucei:  The Native Structure and the Structure in Complex with α-Difluoromethylornithine. Biochemistry 1999, 38 (46), 1517415184,  DOI: 10.1021/bi9915115
    150. 150
      Eckstein, J. W.; Foster, P. G.; Finer-Moore, J.; Wataya, Y.; Santi, D. V. Mechanism-Based Inhibition of Thymidylate Synthase by 5-(Trifluoromethyl)-2’-Deoxyuridine 5′-Monophosphate. Biochemistry 1994, 33 (50), 1508615094,  DOI: 10.1021/bi00254a018
    151. 151
      Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Structural Bioinformatics-Based Design of Selective, Irreversible Kinase Inhibitors. Science 2005, 308 (5726), 13181321,  DOI: 10.1126/science1108367
    152. 152
      Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. A Clickable Inhibitor Reveals Context-Dependent Autoactivation of P90 RSK. Nat. Chem. Biol. 2007, 3 (3), 156160,  DOI: 10.1038/nchembio859
    153. 153
      Carroll, F. A. Perspectives on Structure and Mechanism in Organic Chemistry, 2nd ed.; Wiley-VCH: Weinheim, 2014.
    154. 154
      Shaik, S. S. The .Alpha.- and .Beta.-Carbon Substituent Effect on SN2 Reactivity. A Valence-Bond Approach. J. Am. Chem. Soc. 1983, 105 (13), 43594367,  DOI: 10.1021/ja00351a039
    155. 155
      Lundell, N.; Schreitmüller, T. Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective. Anal. Biochem. 1999, 266 (1), 3147,  DOI: 10.1006/abio.1998.2919
    156. 156
      Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Quantitative Reactivity Profiling Predicts Functional Cysteines in Proteomes. Nature 2010, 468 (7325), 790795,  DOI: 10.1038/nature09472
    157. 157
      Weerapana, E.; Simon, G. M.; Cravatt, B. F. Disparate Proteome Reactivity Profiles of Carbon Electrophiles. Nat. Chem. Biol. 2008, 4 (7), 405407,  DOI: 10.1038/nchembio.91
    158. 158
      Lonsdale, R.; Burgess, J.; Colclough, N.; Davies, N. L.; Lenz, E. M.; Orton, A. L.; Ward, R. A. Expanding the Armory: Predicting and Tuning Covalent Warhead Reactivity. J. Chem. Inf. Model. 2017, 57 (12), 31243137,  DOI: 10.1021/acs.jcim.7b00553
    159. 159
      Allimuthu, D.; Adams, D. J. 2-Chloropropionamide As a Low-Reactivity Electrophile for Irreversible Small-Molecule Probe Identification. ACS Chem. Biol. 2017, 12 (8), 21242131,  DOI: 10.1021/acschembio.7b00424
    160. 160
      Steinmetz, C. G.; Xie, P.; Weiner, H.; Hurley, T. D. Structure of Mitochondrial Aldehyde Dehydrogenase: The Genetic Component of Ethanol Aversion. Structure 1997, 5 (5), 701711,  DOI: 10.1016/S0969-2126(97)00224-4
    161. 161
      Karala, A.-R.; Lappi, A.-K.; Ruddock, L. W. Modulation of an Active-Site Cysteine PKa Allows PDI to Act as a Catalyst of Both Disulfide Bond Formation and Isomerization. J. Mol. Biol. 2010, 396 (4), 883892,  DOI: 10.1016/j.jmb.2009.12.014
    162. 162
      Wang, C.; Abegg, D.; Hoch, D. G.; Adibekian, A. Chemoproteomics-Enabled Discovery of a Potent and Selective Inhibitor of the DNA Repair Protein MGMT. Angew. Chem., Int. Ed. 2016, 55 (8), 29112915,  DOI: 10.1002/anie.201511301
    163. 163
      Boren, B. C.; Narayan, S.; Rasmussen, L. K.; Zhang, L.; Zhao, H.; Lin, Z.; Jia, G.; Fokin, V. V. Ruthenium-Catalyzed Azide–Alkyne Cycloaddition: Scope and Mechanism. J. Am. Chem. Soc. 2008, 130 (28), 89238930,  DOI: 10.1021/ja0749993
    164. 164
      Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Epoxide Electrophiles as Activity-Dependent Cysteine Protease Profiling and Discovery Tools. Chem. Biol. 2000, 7 (8), 569581,  DOI: 10.1016/S1074-5521(00)00014-4
    165. 165
      Willems, L. I.; Jiang, J.; Li, K.-Y.; Witte, M. D.; Kallemeijn, W. W.; Beenakker, T. J. N.; Schröder, S. P.; Aerts, J. M. F. G.; van der Marel, G. A.; Codée, J. D. C.; Overkleeft, H. S. From Covalent Glycosidase Inhibitors to Activity-Based Glycosidase Probes. Chem. - Eur. J. 2014, 20 (35), 1086410872,  DOI: 10.1002/chem.201404014
    166. 166
      Adams, B. T.; Niccoli, S.; Chowdhury, M. A.; Esarik, A. N. K.; Lees, S. J.; Rempel, B. P.; Phenix, C. P. N -Alkylated Aziridines Are Easily-Prepared, Potent, Specific and Cell-Permeable Covalent Inhibitors of Human β-Glucocerebrosidase. Chem. Commun. 2015, 51 (57), 1139011393,  DOI: 10.1039/C5CC03828F
    167. 167
      Singh, G. S. Synthetic Aziridines in Medicinal Chemistry: A Mini-Review. Mini-Rev. Med. Chem. 2016, 16, 892904,  DOI: 10.2174/1389557515666150709122244
    168. 168
      Pitscheider, M.; Mäusbacher, N.; Sieber, S. A. Antibiotic Activity and Target Discovery of Three-Membered Natural Product-Derived Heterocycles in Pathogenic Bacteria. Chem. Sci. 2012, 3, 20352041,  DOI: 10.1039/c2sc20290e
    169. 169
      Lee, M.; Ikejiri, M.; Klimpel, D.; Toth, M.; Espahbodi, M.; Hesek, D.; Forbes, C.; Kumarasiri, M.; Noll, B. C.; Chang, M.; Mobashery, S. Structure–Activity Relationship for Thiirane-Based Gelatinase Inhibitors. ACS Med. Chem. Lett. 2012, 3 (6), 490495,  DOI: 10.1021/ml300050b
    170. 170
      Harshbarger, W.; Miller, C.; Diedrich, C.; Sacchettini, J. Crystal Structure of the Human 20S Proteasome in Complex with Carfilzomib. Structure 2015, 23 (2), 418424,  DOI: 10.1016/j.str.2014.11.017
    171. 171
      Falagas, M. E.; Vouloumanou, E. K.; Samonis, G.; Vardakas, K. Z. Fosfomycin. Clin. Microbiol. Rev. 2016, 29 (2), 321347,  DOI: 10.1128/CMR.00068-15
    172. 172
      Kim, D. H.; Lees, W. J.; Kempsell, K. E.; Lane, W. S.; Duncan, K.; Walsh, C. T. Characterization of a Cys115 to Asp Substitution in the Escherichia Coli Cell Wall Biosynthetic Enzyme UDP-GlcNAc Enolpyruvyl Transferase (MurA) That Confers Resistance to Inactivation by the Antibiotic Fosfomycin. Biochemistry 1996, 35 (15), 49234928,  DOI: 10.1021/bi952937w
    173. 173
      Eschenburg, S.; Priestman, M.; Schönbrunn, E. Evidence That the Fosfomycin Target Cys115 in UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) Is Essential for Product Release. J. Biol. Chem. 2005, 280 (5), 37573763,  DOI: 10.1074/jbc.M411325200
    174. 174
      Porter, N. J.; Christianson, D. W. Binding of the Microbial Cyclic Tetrapeptide Trapoxin A to the Class I Histone Deacetylase HDAC8. ACS Chem. Biol. 2017, 12 (9), 22812286,  DOI: 10.1021/acschembio.7b00330
    175. 175
      Lopus, M.; Smiyun, G.; Miller, H.; Oroudjev, E.; Wilson, L.; Jordan, M. A. Mechanism of Action of Ixabepilone and Its Interactions with the ΒIII-Tubulin Isotype. Cancer Chemother. Pharmacol. 2015, 76 (5), 10131024,  DOI: 10.1007/s00280-015-2863-z
    176. 176
      Carmi, C.; Cavazzoni, A.; Vezzosi, S.; Bordi, F.; Vacondio, F.; Silva, C.; Rivara, S.; Lodola, A.; Alfieri, R. R.; La Monica, S.; Galetti, M.; Ardizzoni, A.; Petronini, P. G.; Mor, M. Novel Irreversible Epidermal Growth Factor Receptor Inhibitors by Chemical Modulation of the Cysteine-Trap Portion. J. Med. Chem. 2010, 53 (5), 20382050,  DOI: 10.1021/jm901558p
    177. 177
      Klüter, S.; Simard, J. R.; Rode, H. B.; Grütter, C.; Pawar, V.; Raaijmakers, H. C. A.; Barf, T. A.; Rabiller, M.; van Otterlo, W. A. L.; Rauh, D. Characterization of Irreversible Kinase Inhibitors by Directly Detecting Covalent Bond Formation: A Tool for Dissecting Kinase Drug Resistance. ChemBioChem 2010, 11 (18), 25572566,  DOI: 10.1002/cbic.201000352
    178. 178
      Gehringer, M.; Forster, M.; Laufer, S. A. Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via Copper-Catalyzed Azide–Alkyne Cycloaddition. ACS Comb. Sci. 2015, 17 (1), 510,  DOI: 10.1021/co500122h
    179. 179
      Forster, M.; Chaikuad, A.; Bauer, S. M.; Holstein, J.; Robers, M. B.; Corona, C. R.; Gehringer, M.; Pfaffenrot, E.; Ghoreschi, K.; Knapp, S.; Laufer, S. A. Selective JAK3 Inhibitors with a Covalent Reversible Binding Mode Targeting a New Induced Fit Binding Pocket. Cell Chem. Biol. 2016, 23 (11), 13351340,  DOI: 10.1016/j.chembiol.2016.10.008
    180. 180
      Rempel, B. P.; Withers, S. G. Covalent Inhibitors of Glycosidases and Their Applications in Biochemistry and Biology. Glycobiology 2008, 18 (8), 570586,  DOI: 10.1093/glycob/cwn041
    181. 181
      Povirk, L. F.; Shuker, D. E. DNA Damage and Mutagenesis Induced by Nitrogen Mustards. Mutat. Res., Rev. Genet. Toxicol. 1994, 318 (3), 205226,  DOI: 10.1016/0165-1110(94)90015-9
    182. 182
      McGregor, L. M.; Jenkins, M. L.; Kerwin, C.; Burke, J. E.; Shokat, K. M. Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes. Biochemistry 2017, 56 (25), 31783183,  DOI: 10.1021/acs.biochem.7b00271
    183. 183
      Ray, S.; Kreitler, D. F.; Gulick, A. M.; Murkin, A. S. The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition. ACS Chem. Biol. 2018, 13 (6), 14701473,  DOI: 10.1021/acschembio.8b00225
    184. 184
      Moynihan, M. M.; Murkin, A. S. Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3-Nitropropionate. Biochemistry 2014, 53 (1), 178187,  DOI: 10.1021/bi401432t
    185. 185
      Krenske, E. H.; Petter, R. C.; Houk, K. N. Kinetics and Thermodynamics of Reversible Thiol Additions to Mono- and Diactivated Michael Acceptors: Implications for the Design of Drugs That Bind Covalently to Cysteines. J. Org. Chem. 2016, 81 (23), 1172611733,  DOI: 10.1021/acs.joc.6b02188
    186. 186
      Krishnan, S.; Miller, R. M.; Tian, B.; Mullins, R. D.; Jacobson, M. P.; Taunton, J. Design of Reversible, Cysteine-Targeted Michael Acceptors Guided by Kinetic and Computational Analysis. J. Am. Chem. Soc. 2014, 136 (36), 1262412630,  DOI: 10.1021/ja505194w
    187. 187
      Smith, S.; Keul, M.; Engel, J.; Basu, D.; Eppmann, S.; Rauh, D. Characterization of Covalent-Reversible EGFR Inhibitors. ACS Omega 2017, 2 (4), 15631575,  DOI: 10.1021/acsomega.7b00157
    188. 188
      Forster, M.; Chaikuad, A.; Dimitrov, T.; Döring, E.; Holstein, J.; Berger, B.-T.; Gehringer, M.; Ghoreschi, K.; Müller, S.; Knapp, S.; Laufer, S. A. Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4-d]Pyrrolo[2,3-b]Pyridine Scaffold. J. Med. Chem. 2018, 61 (12), 53505366,  DOI: 10.1021/acs.jmedchem.8b00571
    189. 189
      Cheung, S. T.; Miller, M. S.; Pacoma, R.; Roland, J.; Liu, J.; Schumacher, A. M.; Hsieh-Wilson, L. C. Discovery of a Small-Molecule Modulator of Glycosaminoglycan Sulfation. ACS Chem. Biol. 2017, 12 (12), 31263133,  DOI: 10.1021/acschembio.7b00885
    190. 190
      Cully, M. Rational Drug Design: Tuning Kinase Inhibitor Residence Time. Nat. Rev. Drug Discovery 2015, 14, 457,  DOI: 10.1038/nrd4673
    191. 191
      Langrish, C. L.; Bradshaw, J. M.; Owens, T. D.; Campbell, R. L.; Francesco, M. R.; Karr, D. E.; Murray, S. K.; Quesenberry, R. C.; Smith, P. F.; Taylor, M. D.; Zhu, J.; Nunn, P. A.; Gourlay, S. G. PRN1008, a Reversible Covalent BTK Inhibitor in Clinical Development for Immune Thrombocytopenic Purpura. Blood 2017, 130, 1052
    192. 192
      Masjedizadeh, M. R.; Gourlay, S. Salts and Solid Form of a Btk Inhibitor. WO2015127310, August 28, 2015.
    193. 193
      Holm, K. J.; Spencer, C. M. Entacapone. Drugs 1999, 58 (1), 159177,  DOI: 10.2165/00003495-199958010-00017
    194. 194
      Mendgen, T.; Steuer, C.; Klein, C. D. Privileged Scaffolds or Promiscuous Binders: A Comparative Study on Rhodanines and Related Heterocycles in Medicinal Chemistry. J. Med. Chem. 2012, 55 (2), 743753,  DOI: 10.1021/jm201243p
    195. 195
      Schneider, T. H.; Rieger, M.; Ansorg, K.; Sobolev, A. N.; Schirmeister, T.; Engels, B.; Grabowsky, S. Vinyl Sulfone Building Blocks in Covalently Reversible Reactions with Thiols. New J. Chem. 2015, 39 (7), 58415853,  DOI: 10.1039/C5NJ00368G
    196. 196
      Siklos, M.; BenAissa, M.; Thatcher, G. R. J. Cysteine Proteases as Therapeutic Targets: Does Selectivity Matter? A Systematic Review of Calpain and Cathepsin Inhibitors. Acta Pharm. Sin. B 2015, 5 (6), 506519,  DOI: 10.1016/j.apsb.2015.08.001
    197. 197
      Cleary, J. A.; Doherty, W.; Evans, P.; Malthouse, J. P. G. Quantifying Tetrahedral Adduct Formation and Stabilization in the Cysteine and the Serine Proteases. Biochim. Biophys. Acta, Proteins Proteomics 2015, 1854, 13821391,  DOI: 10.1016/j.bbapap.2015.07.006
    198. 198
      Sanches, M.; Duffy, N. M.; Talukdar, M.; Thevakumaran, N.; Chiovitti, D.; Canny, M. D.; Lee, K.; Kurinov, I.; Uehling, D.; Al-awar, R.; Poda, G.; Prakesch, M.; Wilson, B.; Tam, V.; Schweitzer, C.; Toro, A.; Lucas, J. L.; Vuga, D.; Lehmann, L.; Durocher, D.; Zeng, Q.; Patterson, J. B.; Sicheri, F. Structure and Mechanism of Action of the Hydroxy–Aryl–Aldehyde Class of IRE1 Endoribonuclease Inhibitors. Nat. Commun. 2014, 5, 4202,  DOI: 10.1038/ncomms5202
    199. 199
      Larraufie, M.-H.; Yang, W. S.; Jiang, E.; Thomas, A. G.; Slusher, B. S.; Stockwell, B. R. Incorporation of Metabolically Stable Ketones into a Small Molecule Probe to Increase Potency and Water Solubility. Bioorg. Med. Chem. Lett. 2015, 25 (21), 47874792,  DOI: 10.1016/j.bmcl.2015.07.018
    200. 200
      Cross, B. C. S.; Bond, P. J.; Sadowski, P. G.; Jha, B. K.; Zak, J.; Goodman, J. M.; Silverman, R. H.; Neubert, T. A.; Baxendale, I. R.; Ron, D.; Harding, H. P. The Molecular Basis for Selective Inhibition of Unconventional MRNA Splicing by an IRE1-Binding Small Molecule. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (15), E869E878,  DOI: 10.1073/pnas.1115623109
    201. 201
      Knoepfel, T.; Furet, P.; Mah, R.; Buschmann, N.; Leblanc, C.; Ripoche, S.; Graus-Porta, D.; Wartmann, M.; Galuba, I.; Fairhurst, R. A. 2-Formylpyridyl Ureas as Highly Selective Reversible-Covalent Inhibitors of Fibroblast Growth Factor Receptor 4. ACS Med. Chem. Lett. 2018, 9 (3), 215220,  DOI: 10.1021/acsmedchemlett.7b00485
    202. 202
      LoPachin, R. M.; Gavin, T. Molecular Mechanisms of Aldehyde Toxicity: A Chemical Perspective. Chem. Res. Toxicol. 2014, 27 (7), 10811091,  DOI: 10.1021/tx5001046
    203. 203
      Caraballo, R.; Dong, H.; Ribeiro, J. P.; Jiménez-Barbero, J.; Ramström, O. Direct STD NMR Identification of β-Galactosidase Inhibitors from a Virtual Dynamic Hemithioacetal System. Angew. Chem., Int. Ed. 2010, 49 (3), 589593,  DOI: 10.1002/anie.200903920
    204. 204
      Buschmann, N.; Fairhurst, R. A.; Knoepfel, T.; Furet, P.; Leblanc, C.; Mah, R.; Kiffe, M.; Graus-Porta, D.; Weiss, A.; Kinyamu-Akunda, J.; Wartmann, M.; Trappe, J.; Gabriel, T. R.; Hofmann, F.; Sellers, W. R. A Reversible Covalent Approach to Selective FGFR4 Inhibition; Book of Abstracts—Frontiers in Medicinal Chemistry Symposium, Jena, 2018; p 26.
    205. 205
      Otto, H.-H.; Schirmeister, T. Cysteine Proteases and Their Inhibitors. Chem. Rev. 1997, 97 (1), 133172,  DOI: 10.1021/cr950025u
    206. 206
      Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson, J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.; Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.; Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. Discovery and Preclinical Profile of Saxagliptin (BMS-477118):  A Highly Potent, Long-Acting, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes. J. Med. Chem. 2005, 48 (15), 50255037,  DOI: 10.1021/jm050261p
    207. 207
      Fleming, F. F.; Yao, L.; Ravikumar, P. C.; Funk, L.; Shook, B. C. Nitrile-Containing Pharmaceuticals: Efficacious Roles of the Nitrile Pharmacophore. J. Med. Chem. 2010, 53 (22), 79027917,  DOI: 10.1021/jm100762r
    208. 208
      Oballa, R. M.; Truchon, J.-F.; Bayly, C. I.; Chauret, N.; Day, S.; Crane, S.; Berthelette, C. A Generally Applicable Method for Assessing the Electrophilicity and Reactivity of Diverse Nitrile-Containing Compounds. Bioorg. Med. Chem. Lett. 2007, 17 (4), 9981002,  DOI: 10.1016/j.bmcl.2006.11.044
    209. 209
      Schmitz, J.; Beckmann, A.-M.; Dudic, A.; Li, T.; Sellier, R.; Bartz, U.; Gütschow, M. 3-Cyano-3-Aza-β-Amino Acid Derivatives as Inhibitors of Human Cysteine Cathepsins. ACS Med. Chem. Lett. 2014, 5 (10), 10761081,  DOI: 10.1021/ml500238q
    210. 210
      Cai, J.; Fradera, X.; van Zeeland, M.; Dempster, M.; Cameron, K. S.; Bennett, D. J.; Robinson, J.; Popplestone, L.; Baugh, M.; Westwood, P.; Bruin, J.; Hamilton, W.; Kinghorn, E.; Long, C.; Uitdehaag, J. C. M. 4-(3-Trifluoromethylphenyl)-Pyrimidine-2-Carbonitrile as Cathepsin S Inhibitors: N3, Not N1 Is Critically Important. Bioorg. Med. Chem. Lett. 2010, 20 (15), 45074510,  DOI: 10.1016/j.bmcl.2010.06.043
    211. 211
      Mac Sweeney, A.; Grosche, P.; Ellis, D.; Combrink, K.; Erbel, P.; Hughes, N.; Sirockin, F.; Melkko, S.; Bernardi, A.; Ramage, P.; Jarousse, N.; Altmann, E. Discovery and Structure-Based Optimization of Adenain Inhibitors. ACS Med. Chem. Lett. 2014, 5 (8), 937941,  DOI: 10.1021/ml500224t
    212. 212
      de Jesus Cortez, F.; Nguyen, P.; Truillet, C.; Tian, B.; Kuchenbecker, K. M.; Evans, M. J.; Webb, P.; Jacobson, M. P.; Fletterick, R. J.; England, P. M. Development of 5N-Bicalutamide, a High-Affinity Reversible Covalent Antiandrogen. ACS Chem. Biol. 2017, 12 (12), 29342939,  DOI: 10.1021/acschembio.7b00702
    213. 213
      Deaton, D. N.; Hassell, A. M.; McFadyen, R. B.; Miller, A. B.; Miller, L. R.; Shewchuk, L. M.; Tavares, F. X.; Willard, D. H.; Wright, L. L. Novel and Potent Cyclic Cyanamide-Based Cathepsin K Inhibitors. Bioorg. Med. Chem. Lett. 2005, 15 (7), 18151819,  DOI: 10.1016/j.bmcl.2005.02.033
    214. 214
      Falgueyret, J.-P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.; Aubin, Y.; Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S. B.; Percival, M. D. Novel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and L. J. Med. Chem. 2001, 44 (1), 94104,  DOI: 10.1021/jm0003440
    215. 215
      Rydzewski, R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S. B.; Bass, K. E.; Wong, D. H. Peptidic 1-Cyanopyrrolidines: Synthesis and SAR of a Series of Potent, Selective Cathepsin Inhibitors. Bioorg. Med. Chem. 2002, 10 (10), 32773284,  DOI: 10.1016/S0968-0896(02)00173-6
    216. 216
      Lainé, D.; Palovich, M.; McCleland, B.; Petitjean, E.; Delhom, I.; Xie, H.; Deng, J.; Lin, G.; Davis, R.; Jolit, A.; Nevins, N.; Zhao, B.; Villa, J.; Schneck, J.; McDevitt, P.; Midgett, R.; Kmett, C.; Umbrecht, S.; Peck, B.; Davis, A. B.; Bettoun, D. Discovery of Novel Cyanamide-Based Inhibitors of Cathepsin C. ACS Med. Chem. Lett. 2011, 2 (2), 142147,  DOI: 10.1021/ml100212k
    217. 217
      Hill, S. V.; Williams, A.; Longridge, J. L. Acid-Catalysed Hydrolysis of Cyanamides: Estimates of Carbodi-Imide Basicity and Tautomeric Equilibrium Constant between Carbodi-Imide and Cyanamide. J. Chem. Soc., Perkin Trans. 2 1984, 0 (6), 10091013,  DOI: 10.1039/p29840001009
    218. 218
      Benson, M. J.; Rodriguez, V.; von Schack, D.; Keegan, S.; Cook, T. A.; Edmonds, J.; Benoit, S.; Seth, N.; Du, S.; Messing, D.; Nickerson-Nutter, C. L.; Dunussi-Joannopoulos, K.; Rankin, A. L.; Ruzek, M.; Schnute, M. E.; Douhan, J. Modeling the Clinical Phenotype of BTK Inhibition in the Mature Murine Immune System. J. Immunol. 2014, 193 (1), 185,  DOI: 10.4049/jimmunol.1302570
    219. 219
      Schwartz, P. A.; Kuzmic, P.; Solowiej, J.; Bergqvist, S.; Bolanos, B.; Almaden, C.; Nagata, A.; Ryan, K.; Feng, J.; Dalvie, D.; Kath, J. C.; Xu, M.; Wani, R.; Murray, B. W. Covalent EGFR Inhibitor Analysis Reveals Importance of Reversible Interactions to Potency and Mechanisms of Drug Resistance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (1), 173178,  DOI: 10.1073/pnas.1313733111
    220. 220
      Zapf, C. W.; Gerstenberger, B. S.; Xing, L.; Limburg, D. C.; Anderson, D. R.; Caspers, N.; Han, S.; Aulabaugh, A.; Kurumbail, R.; Shakya, S.; Li, X.; Spaulding, V.; Czerwinski, R. M.; Seth, N.; Medley, Q. G. Covalent Inhibitors of Interleukin-2 Inducible T Cell Kinase (Itk) with Nanomolar Potency in a Whole-Blood Assay. J. Med. Chem. 2012, 55 (22), 1004710063,  DOI: 10.1021/jm301190s
    221. 221
      Gupta, P.; Wright, S. E.; Kim, S.-H.; Srivastava, S. K. Phenethyl Isothiocyanate: A Comprehensive Review of Anti-Cancer Mechanisms. Biochim. Biophys. Acta, Rev. Cancer 2014, 1846 (2), 405424,  DOI: 10.1016/j.bbcan.2014.08.003
    222. 222
      Hinman, A.; Chuang, H.; Bautista, D. M.; Julius, D. TRP Channel Activation by Reversible Covalent Modification. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (51), 1956419568,  DOI: 10.1073/pnas.0609598103
    223. 223
      van Bladeren, P. J. Glutathione Conjugation as a Bioactivation Reaction. Chem.-Biol. Interact. 2000, 129 (1), 6176,  DOI: 10.1016/S0009-2797(00)00214-3
    224. 224
      Drobnica, L.; Kristián, P.; Augustín, J. The Chemistry of the NCS Group. Cyanates Their Thio Derivatives; Patai, P., Ed.; Wiley: New York, 1977; Part 2, pp 10031221 DOI: 10.1002/9780470771532.ch6 .
    225. 225
      Shibata, T.; Kimura, Y.; Mukai, A.; Mori, H.; Ito, S.; Asaka, Y.; Oe, S.; Tanaka, H.; Takahashi, T.; Uchida, K. Transthiocarbamoylation of Proteins by Thiolated Isothiocyanates. J. Biol. Chem. 2011, 286, 42150,  DOI: 10.1074/jbc.M111.308049
    226. 226
      Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y. Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine. Chem. Res. Toxicol. 2009, 22 (3), 536542,  DOI: 10.1021/tx8003906
    227. 227
      Kumari, V.; Dyba, M. A.; Holland, R. J.; Liang, Y.-H.; Singh, S. V.; Ji, X. Irreversible Inhibition of Glutathione S-Transferase by Phenethyl Isothiocyanate (PEITC), a Dietary Cancer Chemopreventive Phytochemical. PLoS One 2016, 11 (9), e0163821,  DOI: 10.1371/journal.pone.0163821
    228. 228
      Wilson, A. J.; Kerns, J. K.; Callahan, J. F.; Moody, C. J. Keap Calm, and Carry on Covalently. J. Med. Chem. 2013, 56 (19), 74637476,  DOI: 10.1021/jm400224q
    229. 229
      Lewis, S. M.; Li, Y.; Catalano, M. J.; Laciak, A. R.; Singh, H.; Seiner, D. R.; Reilly, T. J.; Tanner, J. J.; Gates, K. S. Inactivation of Protein Tyrosine Phosphatases by Dietary Isothiocyanates. Bioorg. Med. Chem. Lett. 2015, 25 (20), 45494552,  DOI: 10.1016/j.bmcl.2015.08.065
    230. 230
      Cross, J. V.; Foss, F. W.; Rady, J. M.; Macdonald, T. L.; Templeton, D. J. The Isothiocyanate Class of Bioactive Nutrients Covalently Inhibit the MEKK1 Protein Kinase. BMC Cancer 2007, 7 (1), 183,  DOI: 10.1186/1471-2407-7-183
    231. 231
      Mi, L.; Xiao, Z.; Hood, B. L.; Dakshanamurthy, S.; Wang, X.; Govind, S.; Conrads, T. P.; Veenstra, T. D.; Chung, F.-L. Covalent Binding to Tubulin by Isothiocyanates: A Mechanism of Cell Growth Arrest and Apoptosis. J. Biol. Chem. 2008, 283 (32), 2213622146,  DOI: 10.1074/jbc.M802330200
    232. 232
      Ouertatani-Sakouhi, H.; El-Turk, F.; Fauvet, B.; Roger, T.; Le Roy, D.; Karpinar, D. P.; Leng, L.; Bucala, R.; Zweckstetter, M.; Calandra, T.; Lashuel, H. A. A New Class of Isothiocyanate-Based Irreversible Inhibitors of Macrophage Migration Inhibitory Factor. Biochemistry 2009, 48 (41), 98589870,  DOI: 10.1021/bi900957e
    233. 233
      Brown, K. K.; Hampton, M. B. Biological Targets of Isothiocyanates. Biochim. Biophys. Acta, Gen. Subj. 2011, 1810 (9), 888894,  DOI: 10.1016/j.bbagen.2011.06.004
    234. 234
      Pearson, R. J.; Blake, D. G.; Mezna, M.; Fischer, P. M.; Westwood, N. J.; McInnes, C. The Meisenheimer Complex as a Paradigm in Drug Discovery: Reversible Covalent Inhibition through C67 of the ATP Binding Site of PLK1. Cell Chem. Biol. 2018, 25, 11071116,  DOI: 10.1016/j.chembiol.2018.06.001
    235. 235
      Federici, L.; Lo Sterzo, C.; Pezzola, S.; Di Matteo, A. D.; Scaloni, F.; Federici, G.; Caccuri, A. M. Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases. Cancer Res. 2009, 69 (20), 80258034,  DOI: 10.1158/0008-5472.CAN-09-1314
    236. 236
      Erlanson, D. A.; Braisted, A. C.; Raphael, D. R.; Randal, M.; Stroud, R. M.; Gordon, E. M.; Wells, J. A. Site-Directed Ligand Discovery. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (17), 93679372,  DOI: 10.1073/pnas.97.17.9367
    237. 237
      Zong, L.; Bartolami, E.; Abegg, D.; Adibekian, A.; Sakai, N.; Matile, S. Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension. ACS Cent. Sci. 2017, 3 (5), 449453,  DOI: 10.1021/acscentsci.7b00080
    238. 238
      Tjin, C. C.; Otley, K. D.; Baguley, T. D.; Kurup, P.; Xu, J.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. Glutathione-Responsive Selenosulfide Prodrugs as a Platform Strategy for Potent and Selective Mechanism-Based Inhibition of Protein Tyrosine Phosphatases. ACS Cent. Sci. 2017, 3 (12), 13221328,  DOI: 10.1021/acscentsci.7b00486
    239. 239
      Weichert, D.; Gmeiner, P. Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications. ACS Chem. Biol. 2015, 10 (6), 13761386,  DOI: 10.1021/acschembio.5b00070
    240. 240
      Rosenbaum, D. M.; Zhang, C.; Lyons, J. A.; Holl, R.; Aragao, D.; Arlow, D. H.; Rasmussen, S. G. F.; Choi, H.-J.; DeVree, B. T.; Sunahara, R. K.; Chae, P. S.; Gellman, S. H.; Dror, R. O.; Shaw, D. E.; Weis, W. I.; Caffrey, M.; Gmeiner, P.; Kobilka, B. K. Structure and Function of an Irreversible Agonist-Β2 Adrenoceptor Complex. Nature 2011, 469 (7329), 236240,  DOI: 10.1038/nature09665
    241. 241
      Schwalbe, T.; Kaindl, J.; Hübner, H.; Gmeiner, P. Potent Haloperidol Derivatives Covalently Binding to the Dopamine D2 Receptor. Bioorg. Med. Chem. 2017, 25 (19), 50845094,  DOI: 10.1016/j.bmc.2017.06.034
    242. 242
      Liu, Y.; Xie, Z.; Zhao, D.; Zhu, J.; Mao, F.; Tang, S.; Xu, H.; Luo, C.; Geng, M.; Huang, M.; Li, J. Development of the First Generation of Disulfide-Based Subtype-Selective and Potent Covalent Pyruvate Dehydrogenase Kinase 1 (PDK1) Inhibitors. J. Med. Chem. 2017, 60 (6), 22272244,  DOI: 10.1021/acs.jmedchem.6b01245
    243. 243
      Napolitano, L.; Scalise, M.; Koyioni, M.; Koutentis, P.; Catto, M.; Eberini, I.; Parravicini, C.; Palazzolo, L.; Pisani, L.; Galluccio, M.; Console, L.; Carotti, A.; Indiveri, C. Potent Inhibitors of Human LAT1 (SLC7A5) Transporter Based on Dithiazole and Dithiazine Compounds for Development of Anticancer Drugs. Biochem. Pharmacol. 2017, 143, 3952,  DOI: 10.1016/j.bcp.2017.07.006
    244. 244
      Nagy, P. Kinetics and Mechanisms of Thiol–Disulfide Exchange Covering Direct Substitution and Thiol Oxidation-Mediated Pathways. Antioxid. Redox Signaling 2013, 18 (13), 16231641,  DOI: 10.1089/ars.2012.4973
    245. 245
      Go, Y.-M.; Jones, D. P. Thiol/Disulfide Redox States in Signaling and Sensing. Crit. Rev. Biochem. Mol. Biol. 2013, 48 (2), 173181,  DOI: 10.3109/10409238.2013.764840
    246. 246
      García-Santamarina, S.; Boronat, S.; Hidalgo, E. Reversible Cysteine Oxidation in Hydrogen Peroxide Sensing and Signal Transduction. Biochemistry 2014, 53 (16), 25602580,  DOI: 10.1021/bi401700f
    247. 247
      Gupta, V.; Carroll, K. S. Profiling the Reactivity of Cyclic C-Nucleophiles towards Electrophilic Sulfur in Cysteine Sulfenic Acid. Chem. Sci. 2016, 7 (1), 400415,  DOI: 10.1039/C5SC02569A
    248. 248
      Truong, T. H.; Carroll, K. S. Redox Regulation of Epidermal Growth Factor Receptor Signaling through Cysteine Oxidation. Biochemistry 2012, 51 (50), 99549965,  DOI: 10.1021/bi301441e
    249. 249
      Garcia, F. J.; Carroll, K. S. Redox-Based Probes as Tools to Monitor Oxidized Protein Tyrosine Phosphatases in Living Cells. Eur. J. Med. Chem. 2014, 88, 2833,  DOI: 10.1016/j.ejmech.2014.06.040
    250. 250
      Alcock, L. J.; Farrell, K. D.; Akol, M. T.; Jones, G. H.; Tierney, M. M.; Kramer, H. B.; Pukala, T. L.; Bernardes, G. J. L.; Perkins, M. V.; Chalker, J. M. Norbornene Probes for the Study of Cysteine Oxidation. Tetrahedron 2018, 74 (12), 12201228,  DOI: 10.1016/j.tet.2017.11.011
    251. 251
      Poole, T. H.; Reisz, J. A.; Zhao, W.; Poole, L. B.; Furdui, C. M.; King, S. B. Strained Cycloalkynes as New Protein Sulfenic Acid Traps. J. Am. Chem. Soc. 2014, 136 (17), 61676170,  DOI: 10.1021/ja500364r
    252. 252
      Gupta, V.; Carroll, K. S. Rational Design of Reversible and Irreversible Cysteine Sulfenic Acid-Targeted Linear C-Nucleophiles. Chem. Commun. 2016, 52 (16), 34143417,  DOI: 10.1039/C6CC00228E
    253. 253
      Forman, H. J.; Davies, M. J.; Krämer, A. C.; Miotto, G.; Zaccarin, M.; Zhang, H.; Ursini, F. Protein Cysteine Oxidation in Redox Signaling: Caveats on Sulfenic Acid Detection and Quantification. Arch. Biochem. Biophys. 2017, 617, 2637,  DOI: 10.1016/j.abb.2016.09.013
    254. 254
      Gupta, V.; Yang, J.; Liebler, D. C.; Carroll, K. S. Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles. J. Am. Chem. Soc. 2017, 139 (15), 55885595,  DOI: 10.1021/jacs.7b01791
    255. 255
      Holliday, G. L.; Mitchell, J. B. O.; Thornton, J. M. Understanding the Functional Roles of Amino Acid Residues in Enzyme Catalysis. J. Mol. Biol. 2009, 390 (3), 560577,  DOI: 10.1016/j.jmb.2009.05.015
    256. 256
      Platzer, G.; Okon, M.; McIntosh, L. P. PH-Dependent Random Coil 1H, 13C, and 15N Chemical Shifts of the Ionizable Amino Acids: A Guide for Protein PKa Measurements. J. Biomol. NMR 2014, 60 (2–3), 109129,  DOI: 10.1007/s10858-014-9862-y
    257. 257
      Isom, D. G.; Castañeda, C. A.; Cannon, B. R.; Garcia-Moreno, B. E. Large Shifts in PKa Values of Lysine Residues Buried inside a Protein. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (13), 52605265,  DOI: 10.1073/pnas.1010750108
    258. 258
      Hacker, S. M.; Backus, K. M.; Lazear, M. R.; Forli, S.; Correia, B. E.; Cravatt, B. F. Global Profiling of Lysine Reactivity and Ligandability in the Human Proteome. Nat. Chem. 2017, 9 (12), 11811190,  DOI: 10.1038/nchem.2826
    259. 259
      Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.; Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural Determinants of Phosphoinositide 3-Kinase Inhibition by Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine. Mol. Cell 2000, 6 (4), 909919,  DOI: 10.1016/S1097-2765(05)00089-4
    260. 260
      Wymann, M. P.; Bulgarelli-Leva, G.; Zvelebil, M. J.; Pirola, L.; Vanhaesebroeck, B.; Waterfield, M. D.; Panayotou, G. Wortmannin Inactivates Phosphoinositide 3-Kinase by Covalent Modification of Lys-802, a Residue Involved in the Phosphate Transfer Reaction. Mol. Cell. Biol. 1996, 16 (4), 17221733,  DOI: 10.1128/MCB.16.4.1722
    261. 261
      Pettinger, J.; Le Bihan, Y.-V.; Widya, M.; van Montfort, R. L. M.; Jones, K.; Cheeseman, M. D. An Irreversible Inhibitor of HSP72 That Unexpectedly Targets Lysine-56. Angew. Chem., Int. Ed. 2017, 56 (13), 35363540,  DOI: 10.1002/anie.201611907
    262. 262
      Altmeyer, M.; Amtmann, E.; Heyl, C.; Marschner, A.; Scheidig, A. J.; Klein, C. D. Beta-Aminoketones as Prodrugs for Selective Irreversible Inhibitors of Type-1 Methionine Aminopeptidases. Bioorg. Med. Chem. Lett. 2014, 24 (22), 53105314,  DOI: 10.1016/j.bmcl.2014.09.047
    263. 263
      Dahal, U. P.; Gilbert, A. M.; Obach, R. S.; Flanagan, M. E.; Chen, J. M.; Garcia-Irizarry, C.; Starr, J. T.; Schuff, B.; Uccello, D. P.; Young, J. A. Intrinsic Reactivity Profile of Electrophilic Moieties to Guide Covalent Drug Design: N-α-Acetyl-L-Lysine as an Amine Nucleophile. MedChemComm 2016, 7 (5), 864872,  DOI: 10.1039/C6MD00017G
    264. 264
      Anscombe, E.; Meschini, E.; Mora-Vidal, R.; Martin, M. P.; Staunton, D.; Geitmann, M.; Danielson, U. H.; Stanley, W. A.; Wang, L. Z.; Reuillon, T.; Golding, B. T.; Cano, C.; Newell, D. R.; Noble, M. E. M.; Wedge, S. R.; Endicott, J. A.; Griffin, R. J. Identification and Characterization of an Irreversible Inhibitor of CDK2. Chem. Biol. 2015, 22 (9), 11591164,  DOI: 10.1016/j.chembiol.2015.07.018
    265. 265
      Narayanan, A.; Jones, L. H. Sulfonyl Fluorides as Privileged Warheads in Chemical Biology. Chem. Sci. 2015, 6 (5), 26502659,  DOI: 10.1039/C5SC00408J
    266. 266
      Dong, J.; Krasnova, L.; Finn, M. G.; Sharpless, K. B. Sulfur(VI) Fluoride Exchange (SuFEx): Another Good Reaction for Click Chemistry. Angew. Chem., Int. Ed. 2014, 53 (36), 94309448,  DOI: 10.1002/anie.201309399
    267. 267
      Baker, B. R. Irreversible Enzyme Inhibitors. CXLIX. Tissue-Specific Irreversible Inhibitors of Dihydrofolic Reductase. Acc. Chem. Res. 1969, 2 (5), 129136,  DOI: 10.1021/ar50017a001
    268. 268
      Chen, W.; Dong, J.; Plate, L.; Mortenson, D. E.; Brighty, G. J.; Li, S.; Liu, Y.; Galmozzi, A.; Lee, P. S.; Hulce, J. J.; Cravatt, B. F.; Saez, E.; Powers, E. T.; Wilson, I. A.; Sharpless, K. B.; Kelly, J. W. Arylfluorosulfates Inactivate Intracellular Lipid Binding Protein(s) through Chemoselective SuFEx Reaction with a Binding Site Tyr Residue. J. Am. Chem. Soc. 2016, 138 (23), 73537364,  DOI: 10.1021/jacs.6b02960
    269. 269
      Mortenson, D. E.; Brighty, G. J.; Plate, L.; Bare, G.; Chen, W.; Li, S.; Wang, H.; Cravatt, B. F.; Forli, S.; Powers, E. T.; Sharpless, K. B.; Wilson, I. A.; Kelly, J. W. “Inverse Drug Discovery” Strategy To Identify Proteins That Are Targeted by Latent Electrophiles As Exemplified by Aryl Fluorosulfates. J. Am. Chem. Soc. 2018, 140 (1), 200210,  DOI: 10.1021/jacs.7b08366
    270. 270
      James, G. T. Inactivation of the Protease Inhibitor Phenylmethylsulfonyl Fluoride in Buffers. Anal. Biochem. 1978, 86 (2), 574579,  DOI: 10.1016/0003-2697(78)90784-4
    271. 271
      Lively, M. O.; Powers, J. C. Specificity and Reactivity of Human Granulocyte Elastase and Cathepsin G, Porcine Pancreatic Elastase, Bovine Chymotrypsin and Trypsin toward Inhibition with Sulfonyl Flourides. Biochim. Biophys. Acta BBA - Enzymol. 1978, 525 (1), 171179,  DOI: 10.1016/0005-2744(78)90211-5
    272. 272
      Genov, N. C.; Shopova, M.; Boteva, R.; Ricchelli, F.; Jori, G. Intramolecular Distances between Tryptophan Residues and the Active-Site Serine Residue in Alkaline Bacterial Proteinases as Measured by Fluorescence Energy-Transfer Studies. Biochem. J. 1983, 215 (2), 413416,  DOI: 10.1042/bj2150413
    273. 273
      Esch, F. S.; Allison, W. S. Identification of a Tyrosine Residue at a Nucleotide Binding Site in the Beta Subunit of the Mitochondrial ATPase with P-Fluorosulfonyl[14C]-Benzoyl-5′-Adenosine. J. Biol. Chem. 1978, 253, 61006106
    274. 274
      Jörg, M.; Glukhova, A.; Abdul-Ridha, A.; Vecchio, E. A.; Nguyen, A. T. N.; Sexton, P. M.; White, P. J.; May, L. T.; Christopoulos, A.; Scammells, P. J. Novel Irreversible Agonists Acting at the A1 Adenosine Receptor. J. Med. Chem. 2016, 59 (24), 1118211194,  DOI: 10.1021/acs.jmedchem.6b01561
    275. 275
      Yang, X.; Dong, G.; Michiels, T. J. M.; Lenselink, E. B.; Heitman, L.; Louvel, J.; IJzerman, A. P. A Covalent Antagonist for the Human Adenosine A2A Receptor. Purinergic Signalling 2017, 13 (2), 191201,  DOI: 10.1007/s11302-016-9549-9
    276. 276
      Beauglehole, A. R.; Baker, S. P.; Scammells, P. J. Fluorosulfonyl-Substituted Xanthines as Selective Irreversible Antagonists for the A1-Adenosine Receptor. J. Med. Chem. 2000, 43 (26), 49734980,  DOI: 10.1021/jm000181f
    277. 277
      Glukhova, A.; Thal, D. M.; Nguyen, A. T.; Vecchio, E. A.; Jörg, M.; Scammells, P. J.; May, L. T.; Sexton, P. M.; Christopoulos, A. Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity. Cell 2017, 168, 867877,  DOI: 10.1016/j.cell.2017.01.042
    278. 278
      Kitz, R.; Wilson, I. B. Esters of Methanesulfonic Acid as Irreversible Inhibitors of Acetylcholinesterase. J. Biol. Chem. 1962, 237, 32453249
    279. 279
      Moss, D. E.; Berlanga, P.; Hagan, M. M.; Sandoval, H.; Ishida, C. Methanesulfonyl Fluoride (MSF): A Double-Blind, Placebo-Controlled Study of Safety and Efficacy in the Treatment of Senile Dementia of the Alzheimer Type. Alzheimer Dis. Assoc. Disord. 1999, 13 (1), 20,  DOI: 10.1097/00002093-199903000-00003
    280. 280
      Moss, D. E.; Fariello, R. G.; Sahlmann, J.; Sumaya, I.; Pericle, F.; Braglia, E. A Randomized Phase I Study of Methanesulfonyl Fluoride, an Irreversible Cholinesterase Inhibitor, for the Treatment of Alzheimer’s Disease. Br. J. Clin. Pharmacol. 2013, 75 (5), 12311239,  DOI: 10.1111/bcp.12018
    281. 281
      Jones, L. H. Reactive Chemical Probes: Beyond the Kinase Cysteinome. Angew. Chem., Int. Ed. 2018, 57 (30), 92209223,  DOI: 10.1002/anie.201802693
    282. 282
      Mukherjee, H.; Debreczeni, J.; Breed, J.; Tentarelli, S.; Aquila, B.; Dowling, J. E.; Whitty, A.; Grimster, N. P. A Study of the Reactivity of S(VI)–F Containing Warheads with Nucleophilic Amino-Acid Side Chains under Physiological Conditions. Org. Biomol. Chem. 2017, 15 (45), 96859695,  DOI: 10.1039/C7OB02028G
    283. 283
      Lundblad, R. L. Chemical Reagents for Protein Modification, 4th ed.; CRC Press: Boca Raton, 2017.
    284. 284
      Chinthakindi, P. K.; Arvidsson, P. I. Sulfonyl Fluorides (SFs): More Than Click Reagents?. Eur. J. Org. Chem. 2018, 2018 (27–28), 36483666,  DOI: 10.1002/ejoc.201800464
    285. 285
      Wang, N.; Yang, B.; Fu, C.; Zhu, H.; Zheng, F.; Kobayashi, T.; Liu, J.; Li, S.; Ma, C.; Wang, P. G.; Wang, Q.; Wang, L. Genetically Encoding Fluorosulfate-l-Tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo. J. Am. Chem. Soc. 2018, 140 (15), 49954999,  DOI: 10.1021/jacs.8b01087
    286. 286
      Zhao, Q.; Ouyang, X.; Wan, X.; Gajiwala, K. S.; Kath, J. C.; Jones, L. H.; Burlingame, A. L.; Taunton, J. Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes. J. Am. Chem. Soc. 2017, 139 (2), 680685,  DOI: 10.1021/jacs.6b08536
    287. 287
      Becher, I.; Savitski, M. M.; Savitski, M. F.; Hopf, C.; Bantscheff, M.; Drewes, G. Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP. ACS Chem. Biol. 2013, 8 (3), 599607,  DOI: 10.1021/cb3005879
    288. 288
      Baranczak, A.; Liu, Y.; Connelly, S.; Du, W.-G. H.; Greiner, E. R.; Genereux, J. C.; Wiseman, R. L.; Eisele, Y. S.; Bradbury, N. C.; Dong, J.; Noodleman, L.; Sharpless, K. B.; Wilson, I. A.; Encalada, S. E.; Kelly, J. W. A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in Caenorhabditis Elegans. J. Am. Chem. Soc. 2015, 137 (23), 74047414,  DOI: 10.1021/jacs.5b03042
    289. 289
      Grimster, N. P.; Connelly, S.; Baranczak, A.; Dong, J.; Krasnova, L. B.; Sharpless, K. B.; Powers, E. T.; Wilson, I. A.; Kelly, J. W. Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis While Affording a Fluorescent Conjugate. J. Am. Chem. Soc. 2013, 135 (15), 56565668,  DOI: 10.1021/ja311729d
    290. 290
      Dalton, S. E.; Dittus, L.; Thomas, D. A.; Convery, M. A.; Nunes, J.; Bush, J. T.; Evans, J. P.; Werner, T.; Bantscheff, M.; Murphy, J. A.; Campos, S. Selectively Targeting the Kinome-Conserved Lysine of PI3Kδ as a General Approach to Covalent Kinase Inhibition. J. Am. Chem. Soc. 2018, 140 (3), 932939,  DOI: 10.1021/jacs.7b08979
    291. 291
      Gupta, R. C.; Sachana, M.; Mukherjee, I. M.; Doss, R. B.; Malik, J. K.; Milatovic, D. Organophosphates and Carbamates. In Veterinary Toxicology; Gupta, R. C., Ed.; Academic Press, 2018; pp. 495508,  DOI: 10.1016/B978-0-12-811410-0.00037-4 .
    292. 292
      Tamura, T.; Ueda, T.; Goto, T.; Tsukidate, T.; Shapira, Y.; Nishikawa, Y.; Fujisawa, A.; Hamachi, I. Rapid Labelling and Covalent Inhibition of Intracellular Native Proteins Using Ligand-Directed N -Acyl- N -Alkyl Sulfonamide. Nat. Commun. 2018, 9 (1), 1870,  DOI: 10.1038/s41467-018-04343-0
    293. 293
      Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F. Target Discovery in Small-Molecule Cell-Based Screens by in Situ Proteome Reactivity Profiling. Nat. Biotechnol. 2005, 23, 1303,  DOI: 10.1038/nbt1149
    294. 294
      Evans, M. J.; Morris, G. M.; Wu, J.; Olson, A. J.; Sorensen, E. J.; Cravatt, B. F. Mechanistic and Structural Requirements for Active Site Labeling of Phosphoglycerate Mutase by Spiroepoxides. Mol. BioSyst. 2007, 3, 495506,  DOI: 10.1039/b705113a
    295. 295
      Bongard, J.; Lorenz, M.; Vetter, I. R.; Stege, P.; Porfetye, A. T.; Schmitz, A. L.; Kaschani, F.; Wolf, A.; Koch, U.; Nussbaumer, P.; Klebl, B.; Kaiser, M.; Ehrmann, M. Identification of Noncatalytic Lysine Residues from Allosteric Circuits via Covalent Probes. ACS Chem. Biol. 2018, 13 (5), 13071312,  DOI: 10.1021/acschembio.8b00101
    296. 296
      Diethelm, S.; Schafroth, M. A.; Carreira, E. M. Amine-Selective Bioconjugation Using Arene Diazonium Salts. Org. Lett. 2014, 16 (15), 39083911,  DOI: 10.1021/ol5016509
    297. 297
      Tung, C. L.; Wong, C. T. T.; Fung, E. Y. M.; Li, X. Traceless and Chemoselective Amine Bioconjugation via Phthalimidine Formation in Native Protein Modification. Org. Lett. 2016, 18 (11), 26002603,  DOI: 10.1021/acs.orglett.6b00983
    298. 298
      Ritter, E.; Przybylski, P.; Brzezinski, B.; Bartl, F. Schiff Bases in Biological Systems. Curr. Org. Chem. 2009, 13 (3), 241249,  DOI: 10.2174/138527209787314805
    299. 299
      Malátková, P.; Wsól, V. Carbonyl Reduction Pathways in Drug Metabolism. Drug Metab. Rev. 2014, 46 (1), 96123,  DOI: 10.3109/03602532.2013.853078
    300. 300
      Cal, P. M. S. D.; Vicente, J. B.; Pires, E.; Coelho, A. V.; Veiros, L. F.; Cordeiro, C.; Gois, P. M. P. Iminoboronates: A New Strategy for Reversible Protein Modification. J. Am. Chem. Soc. 2012, 134 (24), 1029910305,  DOI: 10.1021/ja303436y
    301. 301
      Adams, J.; Kauffman, M. Development of the Proteasome Inhibitor VelcadeTM (Bortezomib). Cancer Invest. 2004, 22 (2), 304311,  DOI: 10.1081/CNV-120030218
    302. 302
      Bandyopadhyay, A.; McCarthy, K. A.; Kelly, M. A.; Gao, J. Targeting Bacteria via Iminoboronate Chemistry of Amine-Presenting Lipids. Nat. Commun. 2015, 6, 6561,  DOI: 10.1038/ncomms7561
    303. 303
      Bandyopadhyay, A.; Gao, J. Iminoboronate Formation Leads to Fast and Reversible Conjugation Chemistry of α-Nucleophiles at Neutral PH. Chem. - Eur. J. 2015, 21 (42), 1474814752,  DOI: 10.1002/chem.201502077
    304. 304
      Akçay, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.; Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su, Q. Inhibition of Mcl-1 through Covalent Modification of a Noncatalytic Lysine Side Chain. Nat. Chem. Biol. 2016, 12 (11), 931936,  DOI: 10.1038/nchembio.2174
    305. 305
      Harris, T. K.; Turner, G. J. Structural Basis of Perturbed PKa Values of Catalytic Groups in Enzyme Active Sites. IUBMB Life 2002, 53 (2), 8598,  DOI: 10.1080/15216540211468
    306. 306
      Schwans, J. P.; Sunden, F.; Gonzalez, A.; Tsai, Y.; Herschlag, D. Uncovering the Determinants of a Highly Perturbed Tyrosine PKa in the Active Site of Ketosteroid Isomerase. Biochemistry 2013, 52 (44), 78407855,  DOI: 10.1021/bi401083b
    307. 307
      Hett, E. C.; Xu, H.; Geoghegan, K. F.; Gopalsamy, A.; Kyne, R. E.; Menard, C. A.; Narayanan, A.; Parikh, M. D.; Liu, S.; Roberts, L.; Robinson, R. P.; Tones, M. A.; Jones, L. H. Rational Targeting of Active-Site Tyrosine Residues Using Sulfonyl Fluoride Probes. ACS Chem. Biol. 2015, 10 (4), 10941098,  DOI: 10.1021/cb5009475
    308. 308
      Joshi, N. S.; Whitaker, L. R.; Francis, M. B. A Three-Component Mannich-Type Reaction for Selective Tyrosine Bioconjugation. J. Am. Chem. Soc. 2004, 126 (49), 1594215943,  DOI: 10.1021/ja0439017
    309. 309
      Jones, M. W.; Mantovani, G.; Blindauer, C. A.; Ryan, S. M.; Wang, X.; Brayden, D. J.; Haddleton, D. M. Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts. J. Am. Chem. Soc. 2012, 134 (17), 74067413,  DOI: 10.1021/ja211855q
    310. 310
      Ban, H.; Gavrilyuk, J.; Barbas, C. F. Tyrosine Bioconjugation through Aqueous Ene-Type Reactions: A Click-Like Reaction for Tyrosine. J. Am. Chem. Soc. 2010, 132 (5), 15231525,  DOI: 10.1021/ja909062q
    311. 311
      Ban, H.; Nagano, M.; Gavrilyuk, J.; Hakamata, W.; Inokuma, T.; Barbas, C. F. Facile and Stabile Linkages through Tyrosine: Bioconjugation Strategies with the Tyrosine-Click Reaction. Bioconjugate Chem. 2013, 24 (4), 520532,  DOI: 10.1021/bc300665t
    312. 312
      Hatcher, J. M.; Wu, G.; Zeng, C.; Zhu, J.; Meng, F.; Patel, S.; Wang, W.; Ficarro, S. B.; Leggett, A. L.; Powell, C. E.; Marto, J. A.; Zhang, K.; Ngo, J. C. K.; Fu, X.-D.; Zhang, T.; Gray, N. S. SRPKIN-1: A Covalent SRPK1/2 Inhibitor That Potently Converts VEGF from Pro-Angiogenic to Anti-Angiogenic Isoform. Cell Chem. Biol. 2018, 25, 460470,  DOI: 10.1016/j.chembiol.2018.01.013
    313. 313
      Patricelli, M. P.; Nomanbhoy, T. K.; Wu, J.; Brown, H.; Zhou, D.; Zhang, J.; Jagannathan, S.; Aban, A.; Okerberg, E.; Herring, C.; Nordin, B.; Weissig, H.; Yang, Q.; Lee, J.-D.; Gray, N. S.; Kozarich, J. W. In Situ Kinase Profiling Reveals Functionally Relevant Properties of Native Kinases. Chem. Biol. 2011, 18 (6), 699710,  DOI: 10.1016/j.chembiol.2011.04.011
    314. 314
      Sakamoto, H.; Tsukaguchi, T.; Hiroshima, S.; Kodama, T.; Kobayashi, T.; Fukami, T. A.; Oikawa, N.; Tsukuda, T.; Ishii, N.; Aoki, Y. CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant. Cancer Cell 2011, 19 (5), 679690,  DOI: 10.1016/j.ccr.2011.04.004
    315. 315
      Choi, E. J.; Jung, D.; Kim, J.-S.; Lee, Y.; Kim, B. M. Chemoselective Tyrosine Bioconjugation through Sulfate Click Reaction. Chem. - Eur. J. 2018, 24 (43), 1094810952,  DOI: 10.1002/chem.201802380
    316. 316
      Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.; Hett, E. C.; Kyne, R. E.; Robinson, R. P.; Jones, L. H. Covalent Enzyme Inhibition through Fluorosulfate Modification of a Noncatalytic Serine Residue. ACS Chem. Biol. 2017, 12 (8), 20152020,  DOI: 10.1021/acschembio.7b00403
    317. 317
      Crawford, L. A.; Weerapana, E. A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1). Mol. BioSyst. 2016, 12 (6), 17681771,  DOI: 10.1039/C6MB00250A
    318. 318
      Gehringer, M.; Forster, M.; Pfaffenrot, E.; Bauer, S. M.; Laufer, S. A. Novel Hinge-Binding Motifs for Janus Kinase 3 Inhibitors: A Comprehensive Structure–Activity Relationship Study on Tofacitinib Bioisosteres. ChemMedChem 2014, 9 (11), 25162527,  DOI: 10.1002/cmdc.201402252
    319. 319
      Gu, C.; Shannon, D. A.; Colby, T.; Wang, Z.; Shabab, M.; Kumari, S.; Villamor, J. G.; McLaughlin, C. J.; Weerapana, E.; Kaiser, M.; Cravatt, B. F.; van der Hoorn, R. A. L. Chemical Proteomics with Sulfonyl Fluoride Probes Reveals Selective Labeling of Functional Tyrosines in Glutathione Transferases. Chem. Biol. 2013, 20 (4), 541548,  DOI: 10.1016/j.chembiol.2013.01.016
    320. 320
      Ekici, Ö. D.; Paetzel, M.; Dalbey, R. E. Unconventional Serine Proteases: Variations on the Catalytic Ser/His/Asp Triad Configuration. Protein Sci. 2008, 17 (12), 20232037,  DOI: 10.1110/ps.035436.108
    321. 321
      Long, J. Z.; Cravatt, B. F. The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease. Chem. Rev. 2011, 111 (10), 60226063,  DOI: 10.1021/cr200075y
    322. 322
      Lei, J.; Zhou, Y.; Xie, D.; Zhang, Y. Mechanistic Insights into a Classic Wonder Drug—Aspirin. J. Am. Chem. Soc. 2015, 137 (1), 7073,  DOI: 10.1021/ja5112964
    323. 323
      Leney, A. C.; Heck, A. J. R. Native Mass Spectrometry: What Is in the Name?. J. Am. Soc. Mass Spectrom. 2017, 28 (1), 513,  DOI: 10.1007/s13361-016-1545-3
    324. 324
      Li, Z.; Qian, L.; Li, L.; Bernhammer, J. C.; Huynh, H. V.; Lee, J.-S.; Yao, S. Q. Tetrazole Photoclick Chemistry: Reinvestigating Its Suitability as a Bioorthogonal Reaction and Potential Applications. Angew. Chem., Int. Ed. 2016, 55 (6), 20022006,  DOI: 10.1002/anie.201508104
    325. 325
      Zhao, S.; Dai, J.; Hu, M.; Liu, C.; Meng, R.; Liu, X.; Wang, C.; Luo, T. Photo-Induced Coupling Reactions of Tetrazoles with Carboxylic Acids in Aqueous Solution: Application in Protein Labelling. Chem. Commun. 2016, 52 (25), 47024705,  DOI: 10.1039/C5CC10445A
    326. 326
      Mix, K. A.; Raines, R. T. Optimized Diazo Scaffold for Protein Esterification. Org. Lett. 2015, 17 (10), 23582361,  DOI: 10.1021/acs.orglett.5b00840
    327. 327
      Ban, H. S.; Usui, T.; Nabeyama, W.; Morita, H.; Fukuzawa, K.; Nakamura, H. Discovery of Boron-Conjugated 4-Anilinoquinazoline as a Prolonged Inhibitor of EGFR Tyrosine Kinase. Org. Biomol. Chem. 2009, 7 (21), 44154427,  DOI: 10.1039/b909504g
    328. 328
      Woodward, R. B.; Olofson, R. A.; Mayer, H. A New Synthesis of Peptides. J. Am. Chem. Soc. 1961, 83 (4), 10101012,  DOI: 10.1021/ja01465a072
    329. 329
      Martín-Gago, P.; Fansa, E. K.; Winzker, M.; Murarka, S.; Janning, P.; Schultz-Fademrecht, C.; Baumann, M.; Wittinghofer, A.; Waldmann, H. Covalent Protein Labeling at Glutamic Acids. Cell Chem. Biol. 2017, 24, 589597,  DOI: 10.1016/j.chembiol.2017.03.015
    330. 330
      Martín-Gago, P.; Fansa, E. K.; Klein, C. H.; Murarka, S.; Janning, P.; Schürmann, M.; Metz, M.; Ismail, S.; Schultz-Fademrecht, C.; Baumann, M.; Bastiaens, P. I. H.; Wittinghofer, A.; Waldmann, H. A PDE6δ-KRas Inhibitor Chemotype with up to Seven H-Bonds and Picomolar Affinity That Prevents Efficient Inhibitor Release by Arl2. Angew. Chem., Int. Ed. 2017, 56 (9), 24232428,  DOI: 10.1002/anie.201610957
    331. 331
      Tsukiji, S.; Hamachi, I. Ligand-Directed Tosyl Chemistry for in Situ Native Protein Labeling and Engineering in Living Systems: From Basic Properties to Applications. Curr. Opin. Chem. Biol. 2014, 21, 136143,  DOI: 10.1016/j.cbpa.2014.07.012
    332. 332
      Jafari, R.; Almqvist, H.; Axelsson, H.; Ignatushchenko, M.; Lundbäck, T.; Nordlund, P.; Molina, D. M. The Cellular Thermal Shift Assay for Evaluating Drug Target Interactions in Cells. Nat. Protoc. 2014, 9 (9), 21002122,  DOI: 10.1038/nprot.2014.138
    333. 333
      Franken, H.; Mathieson, T.; Childs, D.; Sweetman, G. M. A.; Werner, T.; Tögel, I.; Doce, C.; Gade, S.; Bantscheff, M.; Drewes, G.; Reinhard, F. B. M.; Huber, W.; Savitski, M. M. Thermal Proteome Profiling for Unbiased Identification of Direct and Indirect Drug Targets Using Multiplexed Quantitative Mass Spectrometry. Nat. Protoc. 2015, 10 (10), 15671593,  DOI: 10.1038/nprot.2015.101
    334. 334
      Komissarov, A. A.; Romanova, D. V.; Debabov, V. G. Complete Inactivation of Escherichia Coli Uridine Phosphorylase by Modification of Asp5 with Woodward’s Reagent K. J. Biol. Chem. 1995, 270 (17), 1005010055,  DOI: 10.1074/jbc.270.17.10050
    335. 335
      Qian, Y.; Schürmann, M.; Janning, P.; Hedberg, C.; Waldmann, H. Activity-Based Proteome Profiling Probes Based on Woodward’s Reagent K with Distinct Target Selectivity. Angew. Chem., Int. Ed. 2016, 55 (27), 77667771,  DOI: 10.1002/anie.201602666
    336. 336
      Harlow, K. W.; Switzer, R. L. Chemical Modification of Salmonella Typhimurium Phosphoribosylpyrophosphate Synthetase with 5′-(p-Fluorosulfonylbenzoyl)Adenosine. Identification of an Active Site Histidine. J. Biol. Chem. 1990, 265, 54875493
    337. 337
      Uchida, K.; Stadtman, E. R. Modification of Histidine Residues in Proteins by Reaction with 4-Hydroxynonenal. Proc. Natl. Acad. Sci. U. S. A. 1992, 89 (10), 45444548,  DOI: 10.1073/pnas.89.10.4544
    338. 338
      Yamaguchi, S.; Aldini, G.; Ito, S.; Morishita, N.; Shibata, T.; Vistoli, G.; Carini, M.; Uchida, K. Δ12-Prostaglandin J2 as a Product and Ligand of Human Serum Albumin: Formation of an Unusual Covalent Adduct at His146. J. Am. Chem. Soc. 2010, 132 (2), 824832,  DOI: 10.1021/ja908878n
    339. 339
      Yoshizawa, M.; Itoh, T.; Hori, T.; Kato, A.; Anami, Y.; Yoshimoto, N.; Yamamoto, K. Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands. J. Med. Chem. 2018, 61 (14), 63396349,  DOI: 10.1021/acs.jmedchem.8b00774
    340. 340
      Liu, S.; Widom, J.; Kemp, C. W.; Crews, C. M.; Clardy, J. Structure of Human Methionine Aminopeptidase-2 Complexed with Fumagillin. Science 1998, 282 (5392), 13241327,  DOI: 10.1126/science.282.5392.1324
    341. 341
      Morgen, M.; Jöst, C.; Malz, M.; Janowski, R.; Niessing, D.; Klein, C. D.; Gunkel, N.; Miller, A. K. Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity. ACS Chem. Biol. 2016, 11 (4), 10011011,  DOI: 10.1021/acschembio.5b00755
    342. 342
      Jakob, C. G.; Upadhyay, A. K.; Donner, P. L.; Nicholl, E.; Addo, S. N.; Qiu, W.; Ling, C.; Gopalakrishnan, S. M.; Torrent, M.; Cepa, S. P.; Shanley, J.; Shoemaker, A. R.; Sun, C. C.; Vasudevan, A.; Woller, K. R.; Shotwell, J. B.; Shaw, B.; Bian, Z.; Hutti, J. E. Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315. J. Med. Chem. 2018, 61 (15), 66476657,  DOI: 10.1021/acs.jmedchem.8b00305
    343. 343
      Lin, S.; Yang, X.; Jia, S.; Weeks, A. M.; Hornsby, M.; Lee, P. S.; Nichiporuk, R. V.; Iavarone, A. T.; Wells, J. A.; Toste, F. D.; Chang, C. J. Redox-Based Reagents for Chemoselective Methionine Bioconjugation. Science 2017, 355 (6325), 597602,  DOI: 10.1126/science.aal3316
    344. 344
      Bizet, V.; Hendriks, C. M. M.; Bolm, C. Sulfur Imidations: Access to Sulfimides and Sulfoximines. Chem. Soc. Rev. 2015, 44 (11), 33783390,  DOI: 10.1039/C5CS00208G
    345. 345
      Gong, Y.; Andina, D.; Nahar, S.; Leroux, J.-C.; Gauthier, M. A. Releasable and Traceless PEGylation of Arginine-Rich Antimicrobial Peptides. Chem. Sci. 2017, 8 (5), 40824086,  DOI: 10.1039/C7SC00770A
    346. 346
      Seki, Y.; Ishiyama, T.; Sasaki, D.; Abe, J.; Sohma, Y.; Oisaki, K.; Kanai, M. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 2016, 138 (34), 1079810801,  DOI: 10.1021/jacs.6b06692
    347. 347
      Shibata, Y.; Chiba, M. The Role of Extrahepatic Metabolism in the Pharmacokinetics of the Targeted Covalent Inhibitors Afatinib, Ibrutinib, and Neratinib. Drug Metab. Dispos. 2015, 43 (3), 375384,  DOI: 10.1124/dmd.114.061424
    348. 348
      Scheers, E.; Leclercq, L.; de Jong, J.; Bode, N.; Bockx, M.; Laenen, A.; Cuyckens, F.; Skee, D.; Murphy, J.; Sukbuntherng, J.; Mannens, G. Absorption, Metabolism, and Excretion of Oral 14C Radiolabeled Ibrutinib: An Open-Label, Phase I, Single-Dose Study in Healthy Men. Drug Metab. Dispos. 2015, 43, 289297,  DOI: 10.1124/dmd.114.060061
    349. 349
      Chatterjee, P.; Botello-Smith, W. M.; Zhang, H.; Qian, L.; Alsamarah, A.; Kent, D.; Lacroix, J. J.; Baudry, M.; Luo, Y. Can Relative Binding Free Energy Predict Selectivity of Reversible Covalent Inhibitors?. J. Am. Chem. Soc. 2017, 139 (49), 1794517952,  DOI: 10.1021/jacs.7b08938
    350. 350
      Alberty, R. A.; Hammes, G. G. Application of the Theory of Diffusion-Controlled Reactions to Enzyme Kinetics. J. Phys. Chem. 1958, 62 (2), 154159,  DOI: 10.1021/j150560a005
    351. 351
      Wright, M. H.; Sieber, S. A. Chemical Proteomics Approaches for Identifying the Cellular Targets of Natural Products. Nat. Prod. Rep. 2016, 33 (5), 681708,  DOI: 10.1039/C6NP00001K

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect

This website uses cookies to improve your user experience. By continuing to use the site, you are accepting our use of cookies. Read the ACS privacy policy.

CONTINUE